8/13/2019 AL185A

1/2

HiKaryoXLTM Nutrient Mixture F-10 MediumWith L-Glutamine, FBS, Penicillin, Streptomycin and Sodium bicarbonate

Without Phytohemagglutinin (PHA-M)

1X liquid Karyotyping Medium

Product Code: AL185A

Product description:

HiKaryoXL Nutrient mixture F-10 Medium is a medium

developed for the short term in vitro culture of peripheral

blood lymphocytes for cytogenetic studies. Cytogenetic

studies include metaphase and pro metaphase studies carried

out on lymphocytes to detect chromosomal aberrations

associated with structural and numerical abnormalities.

Lymphocytes come from normal peripheral blood and are

mitotically inactive, hence have to be stimulated with a

mitogen. The most common mitogen used for the stimulation

of cell division in lymphocyte cultures is Phytohemagglutinin

(PHA-M). Phytohemagglutinin is a lectin extract from red

kidney bean (Phaseolus vulgaris). The protein consists of twosubunits, a leucoagglutinin (PHA-L) and an erythroagglutinin

(PHA-E). PHA-M is the mucoprotein form and is used for

stimulation of cell proliferation in lymphocyte culture.

HiKaryoXL Nutrient Mixture F-10 Medium is a

karyotyping medium composed of a basal medium Nutrient

Mixture F-10 and supplemented with L-Glutamine, FBS,

Penicillin, Streptomycin and Sodium bicarbonate. It does not

contain PHA-M, hence if required, has to be added prior too

use.

Composition:

Ingredients mg/LINORGANIC SALTS

Calcium chloride dihydrate 44.100

Copper sulphate pentahydrate 0.0025

Ferric sulphate heptahydrate 0.834

Magnesium sulphate anhydrous 74.730

Potassium chloride 285.000

Potassium dihydrogen phosphate 83.000

Sodium bicarbonate 1200.000

Sodiumchloride 7400.000

Sodium phosphate dibasic anhydrous 153.700

Zinc sulphate heptahydrate 0.0288

AMINO ACIDS

Glycine 7.510

L-Alanine 8.910

L-Arginine hydrochloride 211.000L-Asparagine anhydrous 15.010

L-Aspartic acid 13.300

L-Cystine dihydrochloride 35.130

L-Glutamic acid 14.700

L-Glutamine 146.000

L-Histidine hydrochloride monohydrate 21.000

L-Isoleucine 2.600

L-Leucine 13.100

L-Lysine hydrochloride 29.300

L-Methionine 4.480L-Phenylalanine 4.960

L-Proline 11.500

L-Serine 10.500

L-Threonine 3.570

L-Tryptophan 0.600

L-Tyrosine disodium salt 2.610

L-Valine 3.500VITAMINS

Choline chloride 0.698

D-Biotin 0.024

D-Ca-Pantothenate 0.715

Folic acid 1.320

Niacinamide 0.615

Pyridoxine hydrochloride 0.206

Riboflavin 0.376

Thiamine hydrochloride 1.000

Vitamin B12 1.360

i-Inositol 0.541OTHERS

D-Glucose 1100.000

Fetal Bovine Serum Proprietary

Hypoxanthine sodium salt 4.080

Lipoic acid 0.210

Penicillin Proprietary

Phenol red sodium salt 1.300

Streptomycin Proprietary

Sodium pyruvate 110.000Thymidine 0.730

8/13/2019 AL185A

2/2

Disclaimer:User must ensure suitability of the product(s) in their application prior to use. Products conform solely to the information contained in this and other related

HiMedia Publications. The information contained in this publication is based on our research and development work and is to the best of our knowledge

true and accurate. HiMedia Laboratories Pvt Ltd reserves the right to make changes to specifications and information related to the products at any time.

Products are not intended for human or animal diagnostic or therapeutic use but for laboratory, research or further manufacturing use only, unless

otherwise specified. Statements contained herein should not be considered as a warranty of any kind, expressed or implied, and no liability is accepted for

infringement of any patents.

Revision: 1/ 2010-06

Disclaimer:

User must ensure suitability of the product(s) in their application prior to use. Products conform solely to the information contained in this and other related

HiMedia Publications. The information contained in this publication is based on our research and development work and is to the best of our knowledge

true and accurate. HiMedia Laboratories Pvt Ltd reserves the right to make changes to specifications and information related to the products at any time.

Products are not intended for human or animal diagnostic or therapeutic use but for laboratory, research or further manufacturing use only, unless

otherwise specified. Statements contained herein should not be considered as a warranty of any kind, expressed or implied, and no liability is accepted for

infringement of any patents.

Directions:

1. Add PHA-M to HiKaryoXLTM Medium in required

concentration (5-15g/ml) prior to inoculation of blood.

2. Add whole blood to 10ml of this medium containing

PHA-M in T-25cm2 flasks as per the following

recommendations:

Normal adults - 0.8ml

Infants and children- 0.6ml

Women (during pregnancy/ postpartum) - 1.0ml

Note: If required,.3. Incubate the flasks at 37OC and 5% CO2for 67-72 hours.

4. To determine optimum incubation time i.e. the peak

mitotic index, collect samples at different time intervals

between 48-72 hours.

Note: Peak mitotic index is most commonly observed at

67-72 hrs.

5. Add 100l of 10g/ml of colchicine and incubate for

additional 2 hours.

Note: Incubation time of less than 1 hour might result in

reduced mitotic index

6. Transfer the entire content of the flask to a sterile

centrifuge tube and centrifuge at 800-1000rpm for 10

minutes.

7. Discard the supernatant and resuspend the pellet in 5ml of

hypotonic 0.075M KCl solution and incubate in a water

bath at 37oC for 15-20 minutes.

Note: Add KCl solution drop wise while agitating the

cells

8. Add equal amount of freshly prepared ice cold fixative

(Acetic acid : methanol, 1:3 parts)

9. Centrifuge cells at 800-1000rpm for 10min.

10. Discard the supernatant and again add 5ml of freshly

prepared ice-cold fixative (acetic acid: methanol, 1:3

parts) with constant mixing. Leave the cells at 4oC for

10-15 min.

11. Centrifuge the cells at 1000rpm for 10 minutes.

12. Repeat step no. 10 and 11.

13. Discard the supernatant and resuspend the pellet in 0.2ml

of fresh fixative.

14. Put 1 drop of cell suspension on to a clean, cold slide.

Tilt the slide and let the drop run down the slide as it

spreads and dry the slide rapidly over a hot plate or a

beaker of boiling water.

15. Stain the slides as required.

Materials required but not provided:

HiKaryoXL PHA-M solution 0.1mg/ml (TCL061)

HiKaryoXL PHA-M solution 1mg/ml (TCL071)

HiKaryoXL Colchicine Solution (TCL062) or

HiKaryoXL Colcemid Solution (TCL074)

Potassium Chloride solution 0.075M (TCL040)

Methanol

Acetic Acid,

Giemsa Stain (TCL083)

Quality control:AppearanceOrangish colored, clear solution

pH7.00 -7.60

Osmolality in mOsm/Kg H2O320.00 -360.00

SterilityNo bacterial or fungal growth is observed after 14 days ofincubation, as per USP specification.

Cultural ResponseThe growth promotion capacity of the medium is assessed

qualitatively by analyzing the cells for the morphology andquantitatively by estimating mitotic index.

Storage and shelf life:

Store at -20C. The medium can be aseptically transferred

into smaller aliquots for convenience. Once thawed, use themedium within 30 days.

Avoid exposure to light.Shelf life is 12 months.

Use before expiry date given on the product label.

Following troubleshooting tips can be used in

case of no growth or slow growth:

Cause Solution

Toxic collection

equipment

Check collection equipment. Do a

controlled experiment to rule out

toxicity.

Specimen received in

lithium heparin, EDTA,phenol-preserved heparin,

or other wrong

preservative

Request another specimen if

possible. Wash blood severaltimes before setting up to

increase chances of growth.

Improper shipment or

storage of sample

Check shipment variables: time

passed between collection and

receipt, temperature control,

pressure control, proper pH of

transport medium.

Faulty glassware or plastic

ware

Try another lot or brand of

pipette, flask, Petri dish,

coverglass etc.

PHA- unresponsive culture Try pokeweed mitogen,

Concanavalin A, another

mitogen, or two mitogens. Repeat

the test at a later date if possible.

HiMedia Laboratories Pvt. Ltd. 23 Vadhani Ind. Est., LBS Marg, Mumbai- 400086, India. Customer Care No. 022-4095 1919 Email: techhelp@himedialabs. com