cell culture & transfection - ibian technologies€¦ · breakthrough in mycoplasma detection...

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www.inv ivogen.com/mycoplasma

CELL CULTURE & TRANSFECTION

Mycoplasma Detection and Elimination 7

Reporter Detection Kits 12

Selective Antibiotics 16

Transfection Reagents 19

Induced Pluripotent Stem Cells 20

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Mycoplasma contamination remains a significant problem to the culture of mammalian cells. Mycoplasmas can

cause disastrous effects on eukaryotic cells as they can alter every cellular parameter from proliferation to virus

susceptibility and production leading to unreliable experimental results and potentially unsafe biological products.

Mycoplasmas are the smallest and simplest self-replicating organisms. They lack a rigid cell wall and grow mostly

associated with the mammalian cell membranes. In most cases, there are no signs of mycoplasma contamination.

They cannot be detected by visual inspection and do not cause consistent perceptible changes in a cell culture

such as rapid pH change and medium turbidity. Thus, mycoplasmas can remain undetected in the cell cultures for

long periods.

Mycoplasma Detection

The only way to confirm mycoplasma contamination is by routine testing using special techniques. InvivoGen has developed PlasmoTest™, a

breakthrough in mycoplasma detection for cell cultures. PlasmoTest™ is the first mycoplasma detection kit that uses engineered cells and

therefore can be easily established as a routine procedure in the lab.

Mycoplasma Elimination

Once the mycoplasma contamination has been confirmed, it is usually recommended that the infected cell culture be immediately autoclaved

to prevent the infection from spreading and to use only mycoplasma-free cultures. However, some cell lines are irreplaceable and require an

effective eradication treatment. InvivoGen offers a choice of antimicrobial solutions designed to eliminate and prevent mycoplasma

contaminations. Some are also active against bacteria and/or fungi.

Mycoplasma Detection &

Elimination

• Plasmocin™ - Elimination and Prevention of Mycoplasma

• Plasmocure™ - Elimination of Mycoplasma

• Normocin™ - Prevention Against Mycoplasma, Bacteria and Fungi

• Primocin™ - Prevention Against Mycoplasma, Bacteria and Fungi for Primary Cells

• Fungin™ - Prevention and Elimination of Fungi

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PlasmoTest™ - Mycoplasma Detection

PlasmoTest™ provides a simple, rapid and reliable assay for the visual detection of mycoplasma contamination in cell cultures. This assay is the

first to utilize cells to signal the presence of mycoplasma.

- Simple - Requires only basic cell culture knowledge. No need for specific lab equipment. Results are easily determined with the naked eye or

quantified with a spectrophotometer.

- Rapid - Hands-on time less than 1 hour. Gives results after overnight incubation.

- Versatile - Detects all Mycoplasma and Acholeplasma species known to infect cell cultures, as well as other cell culture contaminants such as bacteria.

- Sensitive - Detects 5.102-5.105 cfu/ml mycoplasmas. No false positive: a positive result indicates the presence of a cell culture contaminant.

- Complete - Contains the Mycoplasma sensor cells and all the reagents needed to perform the assay, including positive and negative controls. Up

to 500 samples can be tested with the kit. To perform further assays, only the reagents need to be reordered.

Principle

PlasmoTest™ features two major constituents: the Mycoplasma sensor cellsand the HEK-Blue™ Detection medium. The Mycoplasma sensor cells detectthe presence of mycoplasmas leading to a color change of the HEK-Blue™

Detection medium. The Mycoplasma sensor cells recognize mycoplasmasthrough Toll-Like Receptor 2 (TLR2), a pathogen recognition receptor. Inthe presence of mycoplasmas, TLR2 initiates a signaling cascade leading tothe activation of NF-kB and other transcription factors. These transcriptionfactors induce the secretion of SEAP (secreted embryonic alkalinephosphatase) in the supernatant which is readily detected by thepurple/blue coloration of the HEK-Blue™ Detection medium.

Key Features

HEK-Blue™-2 cells, the Mycoplasma sensor cells, are engineered HEK293cells. These cells stably express TLR2 and multiple genes from the TLR2pathway and coexpress an optimized SEAP reporter gene, placed underthe control of a promoter inducible by the transcription factors NF-kB andAP-1.

HEK-Blue™ Selection is a solution that combines several selectiveantibiotics. These antibiotics guarantee the persistent expression of thevarious transgenes introduced in HEK-Blue™-2 cells. Furthermore,Normocin™ is included in the kit to protect HEK-Blue™-2 cells from anypotential microbial contamination, whether caused by mycoplasmas,bacteria or fungi.

HEK-Blue™ Detection is a medium specifically designed for the detectionof SEAP. It contains a color substrate that produces a purple/blue colorfollowing its hydrolysis by SEAP (see page 15).

PlasmoTest™ Principle

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PlasmoTest™ Contents

- HEK-Blue™-2 cells (3-5 x 106 cells)- HEK-Blue™ Selection (4 x 2 ml) - Antibiotic mix- Normocin™ (200 mg - 4 x 1 ml)- HEK-Blue™ Detection (2 pouches of 50 ml each)- HEK-Blue™ water (2 x 60 ml) - Positive control- Negative control

Buy the PlasmoTest™ kit once then reorder only the reagents to performfurther assays. The reagents can be purchased individually or together asthe PlasmoTest™ Reagent Kit which includes the following:- HEK-Blue™ Selection (4 x 2 ml) - Antibiotic mix- Normocin™ (4 x 1 ml)- HEK-Blue™ Detection (2 pouches)- HEK-Blue™ water (2 x 60 ml) - Positive control- Negative control

PlasmoTest™ Procedure

1- Collect 500 μl cell culture

supernatant and transfer into amicrotube.

2- Heat samples at 100°C for 15 mins.

3- Prepare HEK-Blue™ Detection byadding 50 ml HEK-Blue™ water to thepowder.

4- Add 50 μl of each sample in a well of

a 96-well plate.

5- Add 50 μl of each control in a well of

a 96-well plate.

6- Prepare HEK-Blue™-2 cell suspensionusing HEK-Blue™ Detection medium.

7- Add 200 μl (~50,000 cells) of cell

suspension to each well containing thesamples or controls.

8- Incubate the plate at 37°C in a CO2

incubator overnight (16-24h).

9- Detect the presence of Mycoplasmawith the naked eye or by reading theOD at 620-655 nm.

PRODUCT QUANTITY CAT. CODE

PlasmoTest™ 1 kit rep-pt2

HEK-Blue™ Detection 2 pouches hb-det1

HEK-Blue™ Selection 5 x 2 ml hb-sel

Normocin™ 500 mg ant-nr-1

PlasmoTest™ Controls 200 tests pt-ctr2

PlasmoTest™ Reagent Kit 500 samples rep-ptrk


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Plasmocin™ - The Mycoplasma Removal Agent

Description

Plasmocin™ is a well-established antimycoplasma reagent. It contains twobactericidal components strongly active against mycoplasmas that allowtheir elimination in only 2 weeks. The first component acts on the proteinsynthesis machinery while the second acts on the DNA replication. Thesetwo specific and separate targets are found only in mycoplasmas andmany other bacteria and are completely absent in eukaryotic cells.

In contrast to other anti-mycoplasma compounds, Plasmocin™ is activeon both free mycoplasmas and intracellular forms. This advantage isconferred by one component of Plasmocin™ which is actively transportedinto mammalian cells. It ensures that following treatment with Plasmocin™

a cell culture is not reinfected by mycoplasmas released from intracellularcompartments of infected cells.

In all animal cell lines tested to date, even at five times the workingconcentration, no apparent adverse effect on cellular metabolism isobserved.

No resistance in liquid cultures of mycoplasmas has ever been identifiedin repeated experiments attempting to measure the mutation rate.Therefore, development of resistant mycoplasma strains is virtuallyeliminated.

Plasmocin™ is also active at low concentrations on a broad range of Grampositive and Gram negative bacteria that are otherwise resistant to themixture of streptomycin and penicillin, and exhibits no toxicity ineukaryotic cells.

Many cell lines infected by mycoplasmas have been successfully treatedwith Plasmocin™, including embryonic stem cells, hybridomas andretrovirus packaging cells.

• Plasmocin™ Treatment To eliminate mycoplasmas use Plasmocin™ (ant-mpt) at25 μg/ml for two weeks in the infected culture.

• Plasmocin™ ProphylacticTo prevent mycoplasma contamination, use Plasmocin™

(ant-mpp) at 2.5 - 5 μg/ml on a regular basis in cell culture.

Plasmocure™ - The Alternative Mycoplasma Removal Agent

Description

In very rare cases, mycoplasmas resistant to Plasmocin™ have beenreported. To eradicate these mycoplasmas, InvivoGen has developed anew antimycoplasma agent called Plasmocure™. Plasmocure™ combinestwo antibiotics that act through different mechanisms of action than thosein Plasmocin™. A two week treatment with Plasmocure™ was foundsufficient to completely eliminate the mycoplasmas. A moderate toxicitycan be observed during the course of the treatment but full recovery ofthe cell line is expected once mycoplasmas are eliminated.

Plasmocure™ is a sterile ready-to-use solution. Simply add to mycoplasmacontaminated cell cultures for 2 weeks at the recommendedconcentration of 50 μg/ml.

Contents and Storage

Plasmocure™ is provided as a colorless solution at a concentration of 100mg/ml. One ml Plasmocure™ is enough to treat 2 L medium. Plasmocure™

is shipped at room temperature. Store at -20°C. Plasmocure™ is stableone year at 4°C or -20°C.


Plasmocin™ is provided as a yellow solution either at a concentration of 25mg/ml (Plasmocin™Treatment) or 2.5 mg/ml (Plasmocin™ Prophylactic). Oneml Plasmocin™ Treatment is enough to treat 1 L medium. Plasmocin™ isshipped at room temperature. Store at -20°C. Plasmocin™ is stable 6 monthsat 4°C and at least one year at -20°C.


Plasmocin™ Treatment 50 mg (2 x 1 ml) ant-mpt

Plasmocin™ Prophylactic 25 mg (5 x 2 ml) ant-mpp


Plasmocure™ 100 mg (1 x 1 ml) ant-pc

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Primocin™ - The Antimicrobial Shield for Primary Cells

Normocin™ - The First Line of Defense for Your Cells

Fungin™ - The Solution for Fungal Contaminations

Description

Primocin™ is an antibiotic formulation specifically designed to protectprimary cell lines from cell culture contaminations. Primocin™ is activeagainst both Gram+ and Gram- bacteria, mycoplasmas and fungi.Primocin™ is the first formulation to offer complete protection againstmicrobial contaminants. There is no need to add Pen/Strep.

Primocin™ is non-toxic to primary cells. It acts on targets found only inmicro-organisms. Bacterial targets are the DNA gyrase and theprokaryotic ribosomal subunits, 30S and 50S. The fungal target isergosterol, a molecule only found in the cell membrane of fungi andyeasts.

Primocin™ is a sterile water soluble solution that can be added directlyto the cell culture medium. The solution is at 50 mg/ml and therecommended working concentration is 100 μg/ml.


Primocin™ is provided as a sterile light yellow solution at a concentrationof 50 mg/ml. Primocin™ is available in 1 ml vials or 20 ml bottles. One mlPrimocin™ is enough to treat 500 ml medium. Primocin™ is shipped atroom temperature and upon receipt should be stored at -20°C.Primocin™ is stable one month at 4°C and 18 months at -20°C.

Description

Normocin™ is an innovative formulation of three antibiotics active againstmycoplasmas, bacteria and fungi. Normocin™ contains two compoundsthat act on mycoplasmas and both Gram+ and Gram- bacteria byblocking DNA and protein synthesis. The third compound eradicatesyeasts and fungi by disrupting ionic exchange through the cell membrane.

The active concentration of Normocin™ (100 μg/ml) displays no

toxicity to the cell line being treated. Normocin™ is used in combinationwith Pen / Strep solutions to broaden the anti-bacterial spectrum.


Normocin™ is provided as a sterile red solution at a concentration of 50mg/ml. Normocin™ is available in 1 ml vials or 20 ml bottles. One mlNormocin™ is enough to treat 500 ml medium. Product is shipped atroom temperature and should be stored at -20°C. Normocin™ is stablethree months at 4°C and 18 months at -20°C.


Fungin™ is provided as a pale yellow solution at a concentration of 10mg/ml. One 1.5 ml vial is able to treat 300 ml to 1.5 liters of mediumdepending on the severity of contamination. Fungin™ is shipped at roomtemperature, store at -20°C. Fungin™ is stable 18 months at 4°C or-20°C.

Description

Fungin™ is a new soluble form of Pimaricin, a polyene introduced in the 1950’sas an anti-fungal agent. This antimycotic compound kills yeasts, molds and fungiby disrupting ionic exchange through the cell membrane. Fungin™ is cell culture tested, and may be added to media containingcommonly used antibacterial agents.

Fungin™ is a highly stable compound. It is shipped at room temperatureand is still active after 6 days at 37°C. Fungin™ is water soluble unlikeAmphotericin B which needs to be dissolved in toxic deoxycholate.Fungin™ exhibits no cytotoxicity to cells being treated, and no deleteriouseffects on cell metabolism. The active concentration of Fungin™ can bestepped up from 10 μg/ml to 50 μg/ml if needed without displaying any

negative effects.


Fungin™ 75 mg (5 x 1.5 ml)

200 mg (1 x 20 ml)

ant-fn-1

ant-fn-2


Normocin™ 500 mg (10 x 1 ml)

1 g (1 x 20 ml)

ant-nr-1

ant-nr-2


Primocin™ 500 mg (10 x 1 ml)

1 g (1 x 20 ml)

ant-pm-1

ant-pm-2


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Reporter Detection Kits

InvivoGen provides two LacZ staining kits:

- LacZ Cell Staining Kit allows you to determine the percentage of

transfected cells expressing the lacZ gene. All reagents are provided toprepare the fixative, staining and rinse solutions. The assay can becompleted in 30 minutes, and the blue precipitate can take from 30minutes to overnight to appear. The LacZ Cell Staining Kit containssufficient reagent to stain one hundred 35 mm plates.

- LacZ Tissue Staining Kit allows the detection of transduced cells

expressing the lacZ gene within fresh or frozen tissues. All reagents areprovided to prepare the buffer, fixative and staining solutions. The stainingof the tissues can be observed in 5 to 24 hours. The LacZ Tissue StainingKit contains sufficient reagent to prepare 100 ml solutions.


The LacZ Cell Staining Kit contains: 10X PBS, X-Gal, PotassiumFerrocyanide, Potassium Ferricyanide, MgCl2 and 10X Fixative Solution. The LacZ Tissue Staining Kit contains: 10X PBS, X-Gal, PotassiumFerrocyanide, Potassium Ferricyanide, MgCl2, Glutaraldehyde, Igepal andNa deoxycholate. Products are shipped at room teperature. Store at 4°C or -20°Caccording to the product label. All reagents are stable for 6 months.

The E. coli lacZ gene encoding b-galactosidase is the classical histochemical reporter gene. b-Galactosidase catalyzes the hydrolysis of X-Gal

producing a blue precipitate that can be easily visualized under a microscope. The LacZ Staining Kits provide simple and convenient methods

for the visual detection of LacZ expression within cells or tissues.

LacZ Reporter Gene System

LacZ Staining Kits

Gene reporter systems are extensively used for the study of eukaryotic gene expression and regulation. Although

they play a significant role in numerous applications, they most frequently serve as indicators of transcriptional

activity in cells. The reporter gene of choice is combined to a promoter sequence and can be subsequently assayed

in vitro or in vivo. An ideal reporter gene is not endogenously expressed by the target cells creating background

signals and can be detected through assays that are easy, sensitive and reliable.

InvivoGen provides two reporter gene systems that can be measured by histochemical staining of cells or tissues:

• b-Galactosidase (LacZ) reporter gene system

• Secreted embryonic alkaline phosphatase (SEAP) reporter gene system

Furthermore, InvivoGen has developed two specific media that allow fast and convenient detection of secreted

AP, called QUANTI-Blue™ and HEK-Blue™ Detection (see pages 14-15).


pSELECT-zeo-LacZ 20 μg psetz-lacz

LacZ Cell Staining Kit 1 kit rep-lz-c

LacZ Tissue Staining Kit 1 kit rep-lz-t

InvivoGen provides the LacZDCpG gene, a humanized and CpG-free

allele of the LacZ gene. This CpG-free gene is ten times more active thanthe wild-type gene in mammalian cells. It can be used for in vitro or invivo applications. The LacZDCpG gene is provided in the pSELECT-zeo-

LacZ plasmid under the control of the EF-1a/HTLV composite promoter.

The pSELECT-zeo-LacZ plasmid is selectable with Zeocin™ in mammaliancells and in bacteria.


The LacZDCpG gene is provided as 20 μg of lyophilized DNA with 4

pouches of E. coli Fast-Media® Zeo (2 TB and 2 Agar, see p45). Plasmid isshipped at room temperature. Store at -20°C for up to one year.

LacZ Reporter Gene

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SEAP Reporter Gene System

Secreted embryonic alkaline phosphatase (SEAP) is a reporter widely used to study promoter activity or gene expression. It is a truncated

form of human placental alkaline phosphatase (PLAP) by deletion of the GPI anchor. Unlike endogenous alkaline phosphatases, PLAP is extremely

heat stable and resistant to the inhibitor L-homoarginine. SEAP is secreted into cell culture supernatant and therefore offers many advantages

over intracellular reporters. It allows to determine reporter activity without disturbing the cells, does not require the preparation of cell

lysates and can be used for kinetic studies.

The SEAP Reporter Assay Kit provides a rapid and convenient methodto determine the levels of SEAP in the culture medium of transfectedcells expressing the seap gene. SEAP catalyzes the hydrolysis ofp-Nitrophenyl phosphate producing a yellow end product that can beread spectrophotometrically at 405 nm.

- Hands-on-time: less than 1 hour

- Obtention of results: 10 to 40 minutes

- Sensitivity: as little as 10-10 g/ml of SEAP

- Concentration range: 1 ng - 1 μg/ml


The SEAP Reporter Assay Kit contains all reagents necessary to measureSEAP activity in 150 samples: 50X Dilution Buffer, 5X Assay Buffer, 100mM L-Homoarginine, tablets of SEAP substrate. Store 50X Dilution Bufferand 5X Assay Buffer at 4°C. Store all other components at -20°C. Allreagents are stable for 6 months when properly stored.

SEAP Reporter Assay Kit


SEAP Reporter Assay Kit 1 kit rep-sap

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InvivoGen provides the secreted embryonic alkaline phosphatase (SEAP)gene in the pSELECT-zeo-SEAP plasmid. It can be used in vivo and in vitroto transfect mammalian cells stably or transiently. The SEAP geneexpression is driven by the EF-1a/HTLV composite promoter that

combines the elongation factor 1 alpha core promoter and the5’untranslated region of the Human T-cell Leukemia Virus. The pSELECT-zeo-SEAP plasmid is selectable with Zeocin™ in both mammalian cellsand bacteria.


The SEAP gene is provided as 20 μg of lyophilized DNA. It is supplied

with 4 pouches of E. coli Fast-Media® Zeo (2 TB and 2 Agar, see p44-45).Plasmid is shipped at room temperature. Store at -20°C for up to oneyear.

SEAP Reporter Gene


pSELECT-zeo-SEAP 20 μg psetz-seap

InvivoGen offers three different methods to determine SEAP activity:

• SEAP Reporter Assay Kit - Detection kit for the quantification of SEAP

• QUANTI-Blue™ - Detection medium for the detection and quantification of any AP

• HEK-Blue™ Detection - Cell culture medium for real-time, one step detection of SEAP

These colorimetric assays are somewhat less sensitive than the chemiluminescent method but are much less expensive and can be performed

using a conventional ELISA plate spectrophotometer.

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QUANTI-Blue™ - Detection and Quantification of Alkaline Phosphatase

QUANTI-Blue™ is a detection medium developed to determine the activity of any alkaline phosphatase (AP) present in a biological sample.

QUANTI-Blue™ offers many advantages over the conventional SEAP Reporter Assay Kit based on the pNPP substrate:

➥ Shorter hands-on-time: no longer than 10 minutes

➥ Simpler preparation: just add water

➥ Allows high-throughput screening

➥ Higher saturation threshold

Key Features

Requires small samples of cell supernatants

Samples of 10 μl are sufficient.

Determine SEAP activity without disturbing cells

The same cell cultures can be repeatedly sampled for kinetic studies orfurther experimentation.

Assay can be completed in 30 min

The enzymatic activity can be detected as early as 20 min after incubationof the samples in QUANTI-Blue™.

Highly sensitive for quantitative measurement

The assay allows to detect low and high levels of SEAP. No need toperform multiple sample dilutions.

Wide dynamic range for accurate measurement

Higher saturation threshold than with pNPP resulting in more significantdifferences between non or low SEAP expression and high SEAPexpression.


QUANTI-Blue™ is provided in a 5- or 10-pouch unit. Each pouch allowsthe preparation of 100 ml of detection medium. Store at roomtemperature. Pouches are stable 12 months at room temperature. Afterpreparation, product is stable 2 weeks at 4°C and 2 months at -20°C.

QUANTI-Blue™ Procedure

* Bulk quantities readily available


QUANTI-Blue™ 5 pouches (5 x 100 ml)

10 pouches (10 x 100 ml)

rep-qb1

rep-qb2

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HEK-Blue™ Detection - Real-Time Detection of SEAP

Key Features

No need to process samples

Preparation of cell lysates or heating of samples are not required.

Determine SEAP activity without disturbing cells

The same cell cultures can be repeatedly sampled for kinetic studies.

Measure the time course of SEAP

Detection of SEAP occurs as the reporter protein is secreted by the cellsgrown in HEK-Blue™ Detection. Being a cell culture medium, in contrast to a simple detection medium such as QUANTI-Blue™,HEK-Blue™ Detection is about 10-fold less sensitive than the former.

Virtually no hands-on time

1- Resuspend HEK-Blue™ Detection by adding water.2- Grow SEAP-expressing cells in HEK-Blue™ Detection.3- Detect SEAP expression with the naked eye or read at OD620-655.


HEK-Blue™ Detection is provided in a 2- or 5-pouch unit. Each pouchallows the preparation of 50 ml of detection medium. Store at roomtemperature. Pouches are stable 12 months at room temperature. Afterpreparation, product is stable 2 weeks at 4°C and 2 months at -20°C.

HEK-Blue™ Detection is a cell culture medium that allows the detection of SEAP as the reporter protein is secreted by the cells. HEK-Blue™

Detection contains all the nutrients necessary for cell growth and a specific SEAP color substrate. The hydrolysis of the substrate by SEAP

produces a purple/blue color that can be easily detected with the naked eye or measured with a spectrophotometer.

➥ One step procedure

➥ Extremely simple to use

➥ Designed for high-throughput screening

HEK-Blue™ Procedure

* Bulk quantities readily available


HEK-Blue™ Detection 2 pouches (2 x 50 ml)

5 pouches (5 x 50 ml)

hb-det1

hb-det2

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Selective Antibiotics

InvivoGen is a leader in the production of selective antibiotics. We manufacture the largest choice of antibiotics

for selection. Our state-of-the-art facilities allow us to produce large quantities of high quality antibiotics with

purity levels exceeding 95%. As we manufacture our products, we are able to offer the best prices on the market.

All our products are provided as cell-culture tested, sterile solutions that are ready-to-use.

High Quality

As all the products are manufactured by InvivoGen, our antibiotics

meet rigorous standards and have passed stringent quality control,

which includes verification of potency, purity and stability using

microbiological and chromatographic methods. This leads to

consistently high quality.

Purity and Stability

All our antibiotics are ultra pure: more than 95% purity for most of

them. Their excellent stability allows for exceptional selection and

reproducible results.

An Excellent Choice of Antibiotics for Selection

in Both Mammalian Cells and E. coli

Matches up to the antibiotic resistance genes carried by InvivoGen

plasmids, built for selection in both mammalian and bacterial cells.

Ready-to-use Cell Culture Tested Solutions

No weighing needed - Our antibiotics are available in solution

filtered to sterility for customer convenience and validated for cell

culture usage.

Endotoxin-free Guaranteed

InvivoGen’s selective antibiotics contain no detectable levels of

endotoxin (<0.125 EU/ml). Many commonly used cell lines, such as

immune cells, express TLR4, the receptor for endotoxins (also

known as lipopolysaccharide (LPS)). The guarantee that our

antibiotics are endotoxin-free ensures that their addition to cell

cultures will not bias the results of your experiments.

Antibiotic Resistance Genes

All InvivoGen’s antibiotics are paired with resistance genes that are

active both in E. coli and mammalian cells. They are available in their

wild-type form in many plasmids provided by InvivoGen, and also as

new synthetic alleles devoid of CpGs in pSELECT and pCpGfree

plasmids (see Chapter 6).

Also Available in Fast-Media®

All our selective antibiotics are available in our ready-made

microwaveable E. coli media Fast-Media®. The antibiotics are at the

appropriate concentration in pre-mixed LB media for selection of

E. coli transformants (see pages 44-45).

Blasticidin

G418 Sulfate

Hygromycin B / HygroGold™

Phleomycin

Puromycin

Zeocin™

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Blasticidin S

Description

Blasticidin S is a peptidyl nucleoside antibiotic isolated from Streptomycesgriseochromogenes. It specifically inhibits protein synthesis in bothprokaryotes and eukaryotes by interfering with the peptide boundformation in the ribosomal machinery. Resistance to blasticidin isconferred by the blasticidin resistance gene from Bacillus cereus (bsr)which encodes a deaminase. Typically, bacteria are sensitive to blasticidinconcentrations of 25-100 μg/ml, and mammalian cells to 1-10 μg/ml.


Blasticidin is provided as a colorless solution at 10 mg/ml. Blasticidin isshipped at room temperature and should be stored at -20°C. Blasticidinis stable at least one year when stored at -20°C.

G418 Sulfate

Description

G418 is an aminoglycoside antibiotic similar in structure to gentamicinB1, produced by Micromonospora rhodorangea. G418 blocks polypeptidesynthesis by inhibiting the elongation step in both prokaryotic andeukaryotic cells. Resistance to G418 is conferred by the neo gene fromTn5 encoding an aminoglycoside 3’-phosphotransferase, APH 3’ II.Selection in mammalian cells is usually achieved in three to seven dayswith concentrations ranging from 400 to 1000 μg/ml. Cells that are

dividing are affected sooner than those that are not.


G418 is provided as a colorless solution at 100 mg/ml. G418 is shippedat room temperature and should be stored at -20°C. G418 is stable forat least one year when stored at -20°C.

Puromycin

Description

Puromycin is an aminonucleoside antibiotic produced by Streptomycesalboniger. It specifically inhibits peptidyl transfer on both prokaryotic andeukaryotic ribosomes. This antibiotic inhibits the growth of Gram positivebacteria and various animal and insect cells. Puromycin can also be usedin some particular conditions for the selection of E. coli transformants.Resistance to puromycin is conferred by the Pac gene encoding apuromycin N-acetyl-transferase (PAC) that was found in a Streptomycesproducer strain. Animal cells are generally sensitive to concentrations from1 to 10 μg/ml.


Puromycin hydrochloride is provided as a colorless solution at 10 mg/ml.Puromycin is shipped at room temperature and should be stored at -20°C.Puromycin is stable at least one year when stored at -20°C.


Blasticidin (solution) 100 mg (5 x 2 ml)

500 mg (25 x 2 ml)

500 mg (1 x 50 ml)

ant-bl-1

ant-bl-5

ant-bl-5b

Blasticidin (powder) 1 g ant-bl-10p


G418 Sulfate 1 g (5 x 2 ml)

5 g (1 x 50 ml)

ant-gn-1

ant-gn-5


Puromycin 100 mg (5 x 2 ml)

500 mg (25 x 2 ml)

ant-pr-1

ant-pr-5

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Hygromycin B

Description

Hygromycin B is an aminoglycoside antibiotic produced by Streptomyceshygroscopicus. It inhibits protein synthesis by interfering with translocationand causing mistranslation at the 70S ribosome. Hygromycin B is effectiveon most bacteria, fungi and higher eukaryotes. Resistance to hygromycin isconferred by the hph gene from E. coli. Hygromycin B is normally used ata concentration of 50-200 μg/ml in mammalian cells and 100 μg/ml in

bacteria.

Two grades of Hygromycin B are available:

• Hygromycin B (purity >85%)

• HygroGold™ (purity >98%)

Phleomycin

Description

Phleomycin is a glycopeptide antibiotic of the bleomycin family, isolatedfrom a mutant strain of Streptomyces verticillus. It binds and intercalatesDNA thus destroying the integrity of the double helix. Phleomycin is activeagainst most bacteria, filamentous fungi, yeast, plant and animal cells. Useof phleomycin is recommended for cells poorly sensitive to Zeocin™, i.e.filamentous fungi and some yeasts. Phleomycin resistance is conferred bythe Sh ble gene from Streptoalloteichus hindustanus which encodes a proteinthat binds to phleomycin, inhibiting its DNA cleavage activity. Typically,phleomycin is used at a concentration of 10 μg/ml for yeasts and 25-150

μg/ml for filamentous fungi.

Zeocin™

Description

Zeocin™ is a formulation of phleomycin D1, a copper-chelatedglycopeptide antibiotic produced by Streptomyces CL990. Zeocin™ causescell death by intercalating into DNA and cleaving it. This antibiotic iseffective on most aerobic cells and is therefore useful for selection in bacteria,eukaryotic microorganisms, plant and animal cells. Resistance to Zeocin™ isconferred by the Sh ble gene product which inactivates Zeocin™ by bindingto the antibiotic. Zeocin™ is used at a concentration of 50-300 μg/ml for

selection in mammalian cells and 25 μg/ml for bacterial selection.


Hygromycin B and HygroGold™ are provided as 100 mg/ml yellowsolutions. HygroGold™ is also provided as a powder. Products are shippedat room temperature. Store at -20°C. Hygromycin B solutions are stablefor at least one year when stored at -20°C.


Zeocin™ is provided as a blue solution at 100 mg/ml. Zeocin™ is shipped atroom temperature and upon receipt should be stored at -20°C. Zeocin™

is stable for at least one year at -20°C.


Phleomycin is provided as a blue solution at 20 mg/ml or as a powder.Phleomycin is shipped at room temperature. Store the solution at -20°Cand the powder at 4°C. Phleomycin is stable for at least one year whenproperly stored.


Phleomycin (solution) 100 mg (5 x 1 ml)

500 mg (25 x 1 ml)

ant-ph-1

ant-ph-5

Phleomycin (powder) 250 mg

500 mg

1 g

ant-ph-2p

ant-ph-5p

ant-ph-10p


Zeocin™ (solution) 1 g (5 x 2 ml)

5 g (25 x 2 ml)

5 g (1 x 50 ml)

ant-zn-1

ant-zn-5

ant-zn-5b

Zeocin™ (powder) 1 g

5 g

ant-zn-1p

ant-zn-5p


Hygromycin B 1 g (5 x 2 ml)

5 g (1 x 50 ml)

ant-hm-1

ant-hm-5

HygroGold™ 1 g (5 x 2 ml)

5 g (1 x 50 ml)

10 g (powder)

ant-hg-1

ant-hg-5

ant-hg-10p

www.inv ivogen.com/transfect ion-reagents18 www.inv ivogen.com/select ive-ant ib iot ics

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DTCPTA

Di-tetradecylphosphoryl-N,N,N-trimethylmethanaminium chloride

DiPPE

1,2-Diphytanoyl-sn-Glycero-3-Phosphoethanolamine

Structure

Perc

enta

ge o

f b-G

al-e

xpre

ssin

g ce

lls1. Floch et al. 1997. Cationic phosphonolipids as non viral vectors for DNA transfectionin hematopoietic cell lines and CD34+ cells. Blood Cells, Molec.& Diseases 23: 69-87.2. Guillaume-Gable et al. 1998. Cationic phosphonolipids as nonviral gene transfer

agents in the lung of mice. Hum. Gene Ther. 9: 2309-2319


LyoVec™ is provided as a lyophilized powder, sterile and packaged in sealed2 ml glass vials (4 or 9 vials). After reconstitution, each vial yields 2 ml oftransfection reagent allowing 40 transfections of 1-4 x 105 cells/well in a 12-well plate. Each order of LyoVec™ comes with 1 vial of control LyoVec™/GFP-LacZ complex. LyoVec™ is shipped at room temperature. Upon receipt, storeat 4°C. LyoVec™ is stable up to 1 year when properly stored.

Transfection efficiencies of LyoVec™: 2 to 8 x 105 cells per 6-well plates weretransfected with 1 μg pVITRO2-GFP/LacZ complexed with 100 μl LyoVec™ in 2 ml

of culture medium containing 10% FBS. LacZ expression was assessed 48h post-transfection. Note: Reagent X is one of the most utilized transfection reagents onthe market. Transfection was performed according to the manufacturer’s protocol.

Features and Benefits

Rapid and Simple-to-use

- Hands-on time: No more than 15 min- No preparation required the day prior transfection

Optimized Procedure

- Works similarly at various lipid/DNA ratios- No optimization of transfection conditions- Soluble in water up to 5X

Maximum Transfection Efficiency

- Transfects with high efficiencies a broad spectrum of mammalian cells,including primary cells and non-adherent cells- Effective for transient and stable transfections

Minimal Cytotoxicity

- Works in the presence of serum- No need to wash cells after transfection

Increased Stability

- Stable over one year when lyophilized- Stable up to six months when rehydrated

Long shelf-life of pDNA/LyoVec Complexes

In contrast with other cationic lipids, plasmid DNA (pDNA)/LyoVec™ complexes remain fully active for transfection for at least twomonths at 4°C. Thus, preparation of large volumes of complexes can bemade and reused repeatedly, saving you time.

LyoVec™ is the first lyophilized cationic lipid-based transfection reagent. The major constituent of LyoVec™ is the phosphonolipid DTCPTA, which

is coupled with DiPPE, a neutral lipid that helps destabilizing membrane bilayers, therefore increasing the in vitro transfection efficiency of LyoVec™.

LyoVec™ New formulation


LyoVec™ 8 ml (160 reactions)

18 ml (360 reactions)

lyec-12

lyec-22

LyoVec™ Procedure

Invivo

LyoV

STEP 1 - LyoVec™ ReconstitutionReconstitute LyoVec™ with 2 ml sterileH2O or PBSNote: Reconstituted LyoVec™ can be storedat 4°C for 6 months.

STEP 2 - Transfection of Adherent andSuspension Cells1-10 μl pDNA (1-3 μg) + 100 μl LyoVec™

-> 20’ at room temperatureNote: Bulk quantities of pDNA/LyoVec™

complexes can be prepared for multiplewells.

1 ml cell suspension (0.25-1.106 cells) +100 μl pDNA/LyoVec™ complexes ->

mix gently. Incubate at 37°C in 5% CO2for 24-72 hours before testing transgeneexpression or applying antibioticselection.

www.inv ivogen.com/transfect ion-reagents 19

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Recently, pioneering work has revealed that terminally differentiated somaticcells can be reprogrammed to generate induced pluripotent stem (iPS) cellsvia overexpression of a defined set of transcription factors. These iPS cellsare morphologically and phenotypically similar to embryonic stem (ES) cellsand thus offer exciting possibilities in stem cell research and regenerativemedicine. Moreover, iPS cells are useful tools for studying the pathogenesisof human disease, for drug discovery and toxicity screening1, 2.

The most widely used set of reprogramming factors, Oct4, Sox2, Klf4 andc-Myc, was identified initially by screening 24 pre-selected factors in mouseembryonic fibroblasts (MEFs) by Takahashi and Yamanaka3. This cocktail oftranscription factors, OSKM, was shown to work for different types ofsomatic cells and for different species, including rhesus monkey4 and humancells5. Direct reprogramming was also achieved by another team who useda partially overlapping combination, Oct4, Sox2, Nanog and Lin28, suggestingthat Oct4 and Sox2 are indispensable whereas Nanog, Lin28, Klf4 andc-Myc are alternative supporting factors6. Subsequent studies havedemonstrated that fewer factors are required; the c-Myc gene, an oncogene,is dispensable (although the efficiency of iPS cell formation is significantlylower)7 and in some cases, such as neural stem cells, expression of only onefactor (Oct4) is sufficient8.

The generation of iPS cells is usually achieved by genetic transduction of thereprogramming genes using retroviral or lentiviral vectors. However, the useof integrating viral vectors represent an obstacle to the therapeutictranslation of iPS cells as this technology can produce insertional mutageniclesions that are potentially tumorigenic. Two recent publications detail theuse of polycistronic lentiviral vectors delivering the OSKM quartet tosomatic cells in a single lentiviral construct reducing the number of genomicinsertions9, 10. Alternative approaches to deliver the reprogramming factorswith minimal or total absence of genetic modifications have been developed.These approaches include the use of LoxP sites and Cre-induced excisionand piggyBac transposon excision of integrated reprogramming vectorsequences11, 12, and the use of an oriP/EBNA1-based episomal vector13.Non-integrating lentiviral vectors may also represent a promising approach14.One possible strategy to entirely replace gene delivery is proteintransduction. Previous studies have demonstrated that various proteins canbe delivered into cells by conjugating them with a short peptide thatmediates cell penetration, such as poly-arginine15. Zhou et al. have designedand purified poly-arginine tagged Oct4, Sox2, Klf4 and c-Myc proteins thatwere found to readily enter cells and translocate into the nucleus16. Afterseveral cycles of protein supplementation, iPS cells were successfullygenerated from MEFs. Using a similar approach, Kim et al. obtainedprotein-induced pluripotent stem cells from human newborn fibroblasts

after several rounds of treatment with cell extracts of HEK293 cell linesexpressing poly-arginine tagged OSKM genes17.

Recent studies have reported an emerging role for microRNAs (miRNAs)in the generation of iPS cells. It has been demonstrated that thereprogramming of iPS cells and subsequent differentiation to lineage specificcells can be observed by monitoring the differential expression ofmiRNAs, small non-coding RNAs that are critical regulators of geneexpression18. Furthermore, the inhibition of tissue-specific miRNAspromotes the formation of iPS cells19.

Direct reprogramming of somatic cells is currently a slow and inefficientprocess, in particular when the c-Myc oncogene is omitted in an effort toreduce tumorigenicity. Several chemicals have recently been reported toeither enhance reprogramming efficiencies or substitute for specificreprogramming factors. Among the reported chemicals, some are knownto affect chromatin modifications while others influence signal transductionpathways. Small molecules that modulate chromatin modifications includeDNA methyltransferase inhibitors (RG108, 5-azacytidine), histonedeacetylase inhibitors (valproic acid, trichostatin A) and a G9a histonemethyltransferase (Bix-01294)20,21,22. Small molecules reported to potentiatereprogramming by targeting signaling pathways include the MEK inhibitorPD035901 and the TGF-beta receptor inhibitor SB43154223.

Among the various reprogramming strategies, the chemical approachcombined to protein transduction may represent the most promising.However, substantial research and development is still required before iPScells are ready for therapeutic applications.

1. Yokoo N. et al., 2009. The effects of cardiovactive drugs on cardiomyocytes derived fromhuman induced pluripotent stem cells. Biochem Biophys Res Commun. 387:482-8. 2. LianQ. et al., 2010. Future perspective of induced pluripotent stem cells for diagnosis, drug

screening and treatment of human diseases. Thromb Haemost. 104(1):39-44. 3. Takahashi

K. & Yamanaka S., 2006. Induction of pluripotent stem cells from mouse embryonic andadult fibroblast cultures by defined factors. Cell. 126(4):663-76. 4. Liu H. et al., 2008.Generation of induced pluripotent stem cells from adult rhesus monkey fibroblasts. CellStem Cell. 3(6):587-90. 5. Takahashi K. et al., 2007. Induction of pluripotent stem cells fromadult human fibroblasts by defined factors. Cell. 131(5):861-72. 6. Yu J. et al., 2007. Inducedpluripotent stem cell lines derived from human somatic cells. Science. 318(5858):1917-20.7. Nakagawa M. et al., 2008. Generation of induced pluripotent stem cells without Myc frommouse and human fibroblasts. Nat Biotechnol. 26(1):101-6. 8. Kim JB. et al., 2009. Oct4-induced pluripotency in adult neural stem cells. Cell. 136(3):411-9. 9. Sommer CA. et al.,2009. Induced pluripotent stem cell generation using a single lentiviral stem cell cassette.Stem Cells. 27(3):543-9. 10. Chang CW. et al., 2009. Polycistronic lentiviral vector for "hitand run" reprogramming of adult skin fibroblasts to induced pluripotent stem cells. StemCells. 27(5):1042-9. 11. Soldner F. et al., 2009. Parkinson's disease patient-derived inducedpluripotent stem cells free of viral reprogramming factors. Cell. 136(5):964-77. 12. Kaji K.et al., 2009. Virus-free induction of pluripotency and subsequent excision of reprogrammingfactors. Nature. 458(7239):771-5. 13. Yu J. et al., 2009. Human induced pluripotent stemcells free of vector and transgene sequences. Science. 324(5928):797-801.14. Sarkis C. et

al., 2008. Non-integrating lentiviral vectors. Curr Gene Ther. 8(6):430-7. Review. 15. OgawaT. et al., 2007. Novel Protein Transduction Method by Using 11R: An Effective New DrugDelivery System for the Treatment of Cerebrovascular Diseases. Stroke, 38: 1354 - 1361.16. Zhou H. et al., 2009. Generation of induced pluripotent stem cells using recombinantproteins. Cell Stem Cell. 4(5):381-4. 17. Kim D. et al., 2009. Generation of human inducedpluripotent stem cells by direct delivery of reprogramming proteins. Cell Stem Cell. 4(6):472-6. 18. Kamata M. et al. 2010. Live cell monitoring of hiPSC generation and differentiationusing differential expression of endogenous microRNAs. PLoS One. 5(7):e11834.19. Mallanna SK. & Rizzino A., 2010. Emerging roles of microRNAs in the control ofembryonic stem cells and the generation of induced pluripotent stem cells. Dev Biol. 344:16-25. 20. Shi Y et al., 2008b. Induction of pluripotent stem cells from mouse embryonicfibroblasts by Oct4 and Klf4 with small-molecule compounds. Cell Stem Cell. 2008 Nov6;3(5):568-74. 21. Huangfu D. et al., 2008. Induction of pluripotent stem cells by definedfactors is greatly improved by small-molecule compounds. Nat Biotechnol. 2008Jul;26(7):795-7. 22. Durcova-Hills G. et al., 2008. Reprogramming primordial germ cells intopluripotent stem cells. PLoS One. 3(10):e3531. 23. Lin T. et al., 2009. A chemical platformfor improved induction of human iPSCs. Nat Methods. 6(11):805-8.

Induced Pluripotent Stem Cells

Induction of pluripotent

stem cells

Somatic cells are obtained fromadult organism.

The reprogramming factors areintroduced into the culturedsomatic cells.

The cells are grown under ES cellsconditions. After 2-3 weeks, iPS cellsemerge.

These induced pluripotent stemcells may be differentiated intovarious cell types for regenerativemedicine applications.

21

Poly-Arginine-HA Tagged Reprogramming Factors

PRODUCTCAT. CODE(HUMAN)

CAT. CODE(MOUSE)

pUNO1-OCT4-11RHA puno1rha-hoct4 puno1rha-moct4

pUNO1-SOX2-11RHA puno1rha-hsox2 puno1rha-msox2

pUNO1-KLF4-11RHA puno1rha-hklf4 puno1rha-mklf4

pUNO1-cMYC-11RHA puno1rha-hmycb puno1rha-mmycb

Description

Poly-arginine-HA tagged reprogramming factors allow the production ofrecombinant cell-penetrating reprogramming factors. They correspond tothe four transcription factors, Oct4, Sox2, Klf4, and c-Myc (OSKM), fused attheir C terminus to a poly-arginine (i.e. 11R) peptide in tandem with 3 motifsof the hemaglutinine (HA) tag. The poly-arginine peptide enables therecombinant proteins to readily enter the cells and have been shown toallow their translocation into the nucleus1, 2. The HA tag is useful for theirdetection by Western blot or their purification by affinity chromatography.

Poly-arginine-HA tagged reprogramming factors are cloned in the pUNO1plasmid within a mammalian expression cassette comprising the EF-1a/HTLV

composite promoter and the SV40 poly adenylation sequence. pUNO1plasmids are selectable in E. coli and mammalian cells with blasticidin.

Application

Poly-arginine-HA tagged reprogramming factors are designed for thegeneration of protein-induced pluripotent cells. Following theirtransfection into mammalian cell lines, such as HEK293, the cell extractscan be used crude to treat the target cells or processed to purifiy thepoly-arginine-HA-tagged proteins on an anti-HA affinity column.


Each pUNO1 plasmid is provided as a lyophilized transformed E. coli strainon a paper disk. Transformed strains are shipped at room temperature andshould be stored at -20°C. Each pUNO1 is provided with 4 pouches ofE. coli Fast-Media® Blas (2 TB and 2 Agar). For more information aboutFast-Media®, see pages 44-45.

1. Zhou H. et al., 2009. Generation of induced pluripotent stem cells using recombinantproteins. Cell Stem Cell. 4(5):381-4. 2. Kim D. et al., 2009. Generation of human inducedpluripotent stem cells by direct delivery of reprogramming proteins. Cell Stem Cell.4(6):472-6.

InvivoGen offers a selection of small molecules that have recently been reported to either enhance reprogramming efficiencies or substitute for specific

reprogramming factors. Among these molecules, some are known to affect chromatin modifications while others influence signal transduction pathways.

Reprogramming Enhancers

PRODUCT DESCRIPTIONWORKING

CONCENTRATIONQUANTITY

CATALOG

CODEREF. INFO

5-Aza-cytidine DNA methyltransferase inhibitor 2 μM 100 mg inh-aza 1, 2 p 157

Bix-01294 G9a histone methyltransferase inhibitor 1 μM 2 mg inh-bix 3, 4 p 157

PD0325901 MEK inhibitor 0.5 μM 2 mg inh-pd32 5, 6 p 156

SB431542 TGF-b receptor inhibitor 2 μM 5 mg inh-sb43 6, 7 p 155

Valproic Acid HDAC inhibitor 2 mM 5 g inh-vpa 2, 8 p 157

1. Mikkelsen TS. et al., 2008. Dissecting direct reprogramming through integrative genomicanalysis. Nature. 454(7200):49-55. 2. Huangfu D. et al., 2008a. Induction of pluripotent stemcells by defined factors is greatly improved by small-molecule compounds. Nat Biotechnol.26(7):795-7. 3. Epsztejn-Litman S. et al., 2008. De novo DNA methylation promoted byG9a prevents reprogramming of embryonically silenced genes. Nat Struct Mol Biol.15(11):1176-83. 4. Shi Y et al., 2008. A combined chemical and genetic approach for thegeneration of induced pluripotent stem cells. Cell Stem Cell. 2(6):525-8. 5. Ying QL. et al.,2008. The ground state of embryonic stem cell self-renewal. Nature. 453(7194):519-23.6. Lin T. et al., 2009. A chemical platform for improved induction of human iPSCs. NatMethods. 6(11):805-8. 7. IInman GJ. et al., 2002. SB-431542 is a potent and specific inhibitor

of transforming growth factor-beta superfamily type I activin receptor-like kinase (ALK)receptors ALK4, ALK5, and ALK7. Mol Pharmacol. 2002 Jul;62(1):65-74. 8 . Huangfu D. et al.,2008b. Induction of pluripotent stem cells from primary human fibroblasts with only Oct4and Sox2. Nat Biotechnol. 26(11):1269-75.


Each product is provided as a solid and shipped at room temperature. Storeat room temperature, 4°C or -20°C according to the product label.

11R

3xHA

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cell culture & transfection - ibian technologies€¦ · breakthrough in mycoplasma detection...

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