Methods 33 (2004) 136–143

www.elsevier.com/locate/ymeth

Transfection of adherent and suspended cells by calcium phosphate

Martin Jordan* and Florian Wurm

LBTC, Laboratory of Cellular Biotechnology, EPFL, Lausanne Switzerland

Received 3 November 2003

Abstract

DNA-calcium phosphate coprecipitates have been used for 30 years as an efficient method to introduce genetic material into cells.

The method involves simple solutions that can be prepared or purchased by the experimentalist. All the numerous variations of the

protocol found in the literature are based on the same principle—a spontaneous precipitation that occurs in supersaturated solu-

tions. When DNA is present during this process, it is readily incorporated into the forming calcium phosphate precipitate. Although

a wide range of conditions will lead to precipitates, high transfection efficiencies are only obtained within a narrow range of op-

timized parameters that assure certain properties of the precipitate. This paper describes several physico-chemical parameters that

are critical to adapt the method to a particular cell line and/or cultivation condition. Examples of protocols that were established

and tested within the authors� laboratory are presented. The article also emphasizes differences between transfections of adherent

and suspended cells.

� 2003 Published by Elsevier Inc.

Keywords: Calcium phosphate; Precipitation; Solubility; Turbidity; CHO; HEK 293; Large scale; Suspension; Transient protein expression;

Osmotic shock

1. Introduction

Calcium phosphate precipitates form either by nu-

cleation or by particle growth. At high relative super-

saturation of calcium and phosphate, nucleation is the

dominant event and creates a large number of new

particles. At low relative supersaturation of these com-

ponents, particle growth predominates. At undersatu-rated conditions, the particles can redissolve. Thus,

calcium phosphate precipitation is a dynamic and re-

versible process, and it can be difficult to achieve its full

potential in routine transfections of mammalian cells.

The calcium ion is undoubtedly a key molecule in the

precipitation process. Removal of calcium through ad-

dition of EGTA rapidly dissolves the precipitate. While

the precipitation occurs spontaneously in the transfec-tion mixture, the size and structure of the precipitate are

strongly influenced by the mixing procedure. DNA is

readily adsorbed onto the precipitate and thereby

changes the characteristics of the particles. When these

* Corresponding author. Fax: +41-21-693-6140.

E-mail address: martin.jordan@epfl.ch (M. Jordan).

1046-2023/$ - see front matter � 2003 Published by Elsevier Inc.

doi:10.1016/j.ymeth.2003.11.011

particles are added to the cells, the pH of the medium

defines the degree of saturation and therefore the fate of

the precipitate, which is normally taken up by cells

within 1 h after contact. Finally, interaction between

complexes and cells is different for static/adherent or

suspended/mixed cultures. These relevant parameters

will be discussed in more detail in the following para-

graphs.

1.1. Role of calcium

Calcium is omnipresent in living organism where it

exists in the mineral form or as an ion. As a mineral,

calcium is found in bones, teeth, and shells. Ionic cal-

cium plays a central role in many cellular processes. The

cytosolic Ca2þ concentration is strictly regulated atlevels clearly below 1 lM. The concentration as well as

the distribution of Ca2þ among intracellular compart-

ments define and control the fate of a cell. For cultured

mammalian cells, calcium is present in the medium.

Here, the calcium concentration is much higher than the

intracellular level but is still kept in the low millimolar

range due to its poor solubility in the presence of

Table 1

Critical parameters affecting the calcium phosphate precipitate

Parameter Effect

Calcium More saturated conditions at higher

concentrations

Phosphate More saturated conditions at higher

concentrations

pH Higher solubility of precipitate at lower pH

DNA DNA strongly interacts with the precipitate

Temperature Lower solubility at higher temperaturesa

Serum It seems to increase the solubility of calcium

and attenuates formation of large precipitates

CO2 Affects the pH and can be incorporated into

the precipitate as CO2�3

a This unusual feature distinguishes calcium phosphate from other

salts.

M. Jordan, F. Wurm / Methods 33 (2004) 136–143 137

phosphate or carbonate ions. Calcium spontaneouslyforms microprecipitates at concentrations around

10mM in standard culture media [1]. Such precipitates

can be observed directly or their appearance is indicated

indirectly by improved transfection efficiencies. Positive

effects of calcium are reported for different methods such

as adenovirus-mediated gene transfer [2,3], cationic

polymers [4], cationic liposomes [5], or cationic proteins

[4]. Enhancing properties of calcium are attributed tothe fusogenic effect of microprecipitates, but the exact

mechanism is still unknown. For the calcium phosphate

technique, the elevated calcium concentration (12mM)

during the transfection might lead to de novo formation

of microprecipitates, which may have a positive effect on

the transfection efficiency. Whether such microprecipi-

tates are formed and whether they are relevant to the

transfection remain open questions.

1.2. Solubility of the precipitate

Once the precipitate has been formed, it can be dif-

ficult to keep its characteristics constant during trans-

fection. In contrast to cationic polymers or other

transfection reagents that are chemically stable under

physiological conditions, calcium phosphate precipitatescontinuously undergo changes that depend on the rela-

tive supersaturation of the solution. Since this can be

affected in many different ways, optimization of the

transfection and troubleshooting are not easy tasks.

The main factors determining the supersaturation are

the concentrations of calcium and phosphate [1,6–8].

Increasing either of them causes a higher degree of su-

persaturation or decreases the solubility of the precipi-tate. The concentrations of both ions are defined when

the precipitation is initiated. Two things should be noted

in this context. First, calcium ions are present in vaste

excess. Therefore, the ongoing precipitation leads to a

depletion of phosphate that stops the whole process

when the solution is no longer supersaturated. At this

point, the calcium ion concentration is still close to its

initial value. Secondly, one has to admit that thechemical composition of ‘‘Ca(PO4)i’’ is not known,

though hydroxyapatite is frequently mentioned in this

context. In fact, several distinctive types of calcium

phosphates do exist [9]. They all have low solubility and

thus could represent the precipitate. Whereas the rele-

vance of the calcium phosphate crystal type for trans-

fections has to be substantiated, it is conceivable that a

precipitate with a different chemical composition isachieved when the solubility is changed.

While the calcium and phosphate concentrations are

reproducible when preparing new transfection solutions,

other factors can be more difficult to control. The pH of

transfection buffers is a key factor that has to be care-

fully controlled [6,10–12]. It influences the solubility of

phosphate by defining the ratios between H2PO�4 ,

HPO2�4 , and the highly insoluble PO3�

4 . For a solutionof 1.4mM phosphate at pH 7.05, as typically used for

transfection (solution B as defined below), one can ex-

pect concentrations of 0.82mM H2PO�4 , 0.58mM

HPO2�4 , and 8.2� 10�6 mM PO3�

4 . Finally, the solubility

is affected by the temperature [13], the DNA concen-

tration [11], and impurities from the chemicals and

DNA [14].

Adding the precipitate to the culture medium resultsin a 10-fold dilution of the calcium concentration. From

this point, the fate of the precipitate depends on addi-

tional factors such as the serum concentration in the

medium, the CO2 saturation of the medium, and cellular

activity. Furthermore, conditions change during the

transfection period (e.g., a decreasing pH value). Any

change can affect the precipitate depending on the sol-

ubility, the particle size, the composition, and the sur-face structure. Since mathematical models are not

available, our concept of the precipitate is based on in-

terpretations of empirical experiments. Table 1 sum-

marizes parameters that change the solubility and/or

affect the precipitation.

1.3. Size of the precipitated particles

The size of the precipitate defines the capacity to bind

DNA and modulates transfection efficiency and toxic

side effects. Although the actual size cannot be truly

controlled, increasing the phosphate concentration fa-

vors larger precipitates (particles with a diameter well

above 1 lm). Such large particles may be agglomerated

nanocrystals rather than individual crystals. The pres-

ence of DNA seems to influence agglomeration [11].Depending on the experimental conditions, agglomer-

ates appear within minutes to hours. A freshly prepared

mixture inevitably becomes inefficient for transfections

when particles become too large (see Fig. 2). Thus, the

time of precipitate formation is critical [7]. Optimal

timing can be affected by any of the parameters men-

tioned in Table 1 [15].

138 M. Jordan, F. Wurm / Methods 33 (2004) 136–143

Having this in mind, we defined our transfectionprotocol rather strictly, but when executed by different

individuals, we still observed significant variation. To

our surprise, it turned out that the mode of mixing so-

lution A with solution B had measurable consequences

on the transfection efficiency. Unfortunately, the mixing

procedure is difficult to define. The volumes of mixing as

well as the speed of mixing need to be considered.1 Even

if not mentioned explicitly, the literature recognizesmixing issues. It is reported that 1ml taken from a 10ml

mixture transfects better than 1ml that was mixed sep-

arately2 [16]. The most fundamental mixing issue is the

speed of mixing. Mixing can be divided into two cate-

gories—‘‘dropwise addition’’ [17–20] and ‘‘fast mixing’’

[7,11–13]. Transfection solutions and/or incubation pe-

riods that work well for one mode are not suitable for

the other [21]. Altogether, there is enough evidence tosupport the view that mixing affects the properties of the

precipitate. The turbidity test (see Section 2.3) is a sen-

sitive tool for addressing the issue of mixing. Repro-

ducible precipitates can be achieved by considering all

these factors and by always mixing in the same way.

1.4. DNA concentration

By varying the DNA concentration in the transfec-

tion cocktail, several authors found a sharp optimum

for protein expression [8,10,11,22]. It can be assumed

that this is due to a physico-chemical effect of the neg-

atively charged DNA that directly influences the for-

mation of the precipitate [23]. At lower concentrations,

DNA�s positive effect does not achieve its full potential,

while at high concentrations the DNA can be detri-mental to the precipitation. Efficient precipitates are

obtained around 25 lgDNA/ml. Using only small

amounts of the DNA of interest and variable amounts

of a nonspecific carrier DNA leads to a similar dose–

response curve [6,24]. Carrier DNA can be either chro-

mosomal (e.g., salmon sperm or calf thymus) or plasmid

DNA, either an empty vector or one carrying a

transgene. We noticed that RNA can also have a carriereffect [25].

The use of carrier DNA has several advantages. It

permits a reduction in the amount of vector DNA in the

transfection. This is important if two or more vectors

are transfected at the same time. Second, cell specific

expression can be reduced to more physiological levels

by decreasing the amount of the transgene vector to a

few percent of the total DNA [26,27].The quality of DNA is another issue. Both CsCl-

purified DNA and DNA recovered from commercially

1 Pipette tips, pipetting speed, angle, force, etc., define the whole

process of mixing that occurs in a matter of seconds.2 According to our experience, this observation is a typical mixing

issue.

available columns are sufficiently pure for transfection.In fact, we have observed that crude DNA was heavily

contaminated with RNA and/or endotoxins transfected

as well as control DNA [28]. On the other hand, we

occasionally see that certain batches of plasmid DNA

simply do not work. Though the interfering components

have not been identified, mixing such DNA with a

normal batch of DNA can attenuate its negative effect

(unpublished data).

1.5. pH of the culture medium

The pH of the medium has not only physiological

effects on the cells, but it also defines the relative su-

persaturation during the transfection. Diluting the pre-

cipitate into the culture medium generally stabilizes the

particles [29], but further maturation of the precipitatewill occur [30,31]. The maturation process is probably

highly complex since different medium components in-

teract with or are incorporated into the existing parti-

cles. Calcium ions from the precipitate can be partially

substituted by Mg2þ, Pb2þ, Zn2þ, etc., and phosphate

ions can be replaced by negative ions such as carbonate.

The latter can be either derived from the CO2 produced

by the cells or from the NaHCO3 added to the mediumas a natural pH buffer. In both cases, the resulting CO2�

3

concentration is related to the pH, which depends on the

liquid/gas equilibrium of CO2, the cell density, and the

accumulation of acidic compounds such as lactate. Al-

together, the cell density, the culture volume, and the

cultivation system affect the pH, thereby influencing the

transfection. To avoid strong pH fluctuations, the ad-

dition of a second pH buffer such as Hepes to the culturemedium is recommended.

1.6. Osmotic shocks

At the end of the transfection period (usually 1–5 h),

free-floating precipitates are removed through a medium

exchange3 that also acts to reset the calcium concen-

tration. At this time, an osmotic shock is frequentlyapplied to boost the expression. Apparently, the osmotic

shock is superfluous for certain cells such as the easily

transfected HEK 293 cells. However, CHO cells can

only be transfected with reasonable efficiency when ap-

plying an osmotic shock. The most frequently used

agents for this purpose are glycerol and DMSO [8,32–

34]. The concentration of the agent (5–20%) and the

duration of the shock (1–10min) have to be adapted foreach cell line to minimize potential toxic effects.

The osmotic shock appears to act on cells that have

already taken up the DNA. Studies using fluorescently

labeled DNA demonstrated that the glycerol shock does

not affect the general uptake of DNA into the cells [35].

3 Particles sticking on the cell surface will not be removed.

Fig. 2. Adherent CHO cells covered with fine (left), medium (middle),

and large (right) particles. The number of particles per cell and the

transfection efficiency decrease with increased particle size.

M. Jordan, F. Wurm / Methods 33 (2004) 136–143 139

This fits with our recent observation that one can re-move precipitates sticking to the cell surface with a brief

EGTA treatment prior to the osmotic shock without

impairing its beneficial effect. Moreover, using time

lapse video imaging to observe individual CHO cells

during transfection experiments, we found that expres-

sion of enhanced green fluorescent protein (EGFP) be-

came apparent shortly after the glycerol treatment

(Fig. 1). The overall frequency of positive cells wasabout 30%. By analyzing the data more carefully, it was

noticed that mitosis was another prerequisite for EGFP

expression. For most of the positive cells, including the

late ones, EGFP was detectable 2–4 h after mitosis.

Prolonged time intervals between osmotic shock and

mitosis dramatically reduced the frequency of EGFP

positive cells. These data support the idea that the

osmotic shock favors the escape of DNA from theendosomal/lysosomal compartment. Once in the cyto-

plasm, the DNA still has to overcome the nuclear

membrane. If not, the DNA will rapidly be degraded by

cytosolic nucleases [36,37]. Video time lapse data fit well

with earlier studies that link high transfectability of cells

with their cell cycle phase [38].

1.7. Adherent versus suspended cells

Principal differences between adherent/static and

mixed/suspended cultures require adaptation of the

transfection protocol. A static culture is de facto a het-

erogeneous system where cells are densely populated at

the plastic surface while the bulk of the medium is de-

prived of cells. In addition, strong vertical gradients

exist for certain key molecules since their transport de-pends on diffusion. Obviously, most transfection com-

plexes added to the medium will not contact cells.

Sufficient contacts will only occur if complexes are

Fig. 1. The time period between glycerol shock and the first appearance

of detectable EGFP following transfection of adherent CHO cells. A

total of 80 positive CHO cells were analyzed. Considering that detec-

tion of EGFP depends on transcription, translation, and maturation of

EGFP—altogether taking about 2 h—the role of the osmotic shock for

DNA entry into the nucleus becomes evident (data courtesy of Dr. F.

Grosjean).

present at high concentrations or if they are big enough

to settle down onto the cells. While a minimal particle

size is needed for gravitational settling, particles that are

too large are taken up with a lower efficiency than

smaller particles. Covering cells with numerous smallparticles instead of a few larger ones increases the

chances that at least one particle will succeed in trans-

ferring its DNA into the nucleus (Fig. 2). We estimate

that each particle, depending on its actual size, can bind

or incorporate 100–10,000 plasmid molecules. Thus, a

single particle delivering its DNA into the nucleus can

be sufficient to transfect the cell.

Suspended cells are constantly mixed, assuring a ho-mogeneous distribution of cells and precipitates. In this

situation, settling is not a relevant issue. As soon as

particles are added to the culture, they will interact with

cells. What counts is the affinity between both compo-

nents. As cells are negatively charged, they preferentially

interact with positively charged particles, independent of

particle size. The latter still might be important for

events occurring during the internalization procedure.A peculiarity of HEK 293 cells cultivated in suspen-

sions is their tendency to grow as aggregates. When

using suspension adapted HEK 293 cells with proper

media (e.g., calcium-reduced media [39]), cell aggrega-

tion is no longer an obstacle. In the presence of the

precipitate and high calcium concentrations, cells will

form aggregates. This does not seem to hinder DNA

uptake since the frequency of positive cells within suchaggregates is not reduced [43].

2. Description of method

2.1. Preparing transfection solutions

Two solutions, one for calcium and the other forphosphate, are needed for transfections. Solution A

140 M. Jordan, F. Wurm / Methods 33 (2004) 136–143

contains 250mM calcium chloride in pure water. Solu-tion B contains 1.4mM phosphate (sodium salt of

H2PO�4 or HPO2�

4 ), 140mM sodium chloride, and

50mM Hepes. The pH of solution B is adjusted at room

temperature to 7.05 using NaOH or HCl. Sterile, ready

to use solutions can be stored at room temperature for

years. Using closed 50ml centrifugation tubes we did

not observe any decline in performance upon storage for

6 months at temperatures ranging from )20 to 37 �C.We routinely keep our solutions in tight 50ml poly-

propylene tubes at room temperature.

It is recommended to compare new batches of

transfection solutions with reference solutions that are

known to work well in transfection. If no reference so-

lutions are available, then it is worth doing a turbidity

test (see below) and a transfection. For both tests, the

variation of at least one key parameter such as phos-phate concentration is recommended. At low concen-

trations of phosphate, no precipitate is detected and no

transfection occurs (Fig. 3). Phosphate levels of 0.5–

1mM are sufficient to coprecipitate DNA, yielding the

best expression levels following transfection (Fig. 3).

Above 1mM phosphate, the precipitate becomes coarser

and expression levels drop (Fig. 3). For these tests, a

simple approach to changing the concentration ofphosphate without modifying solution B is to mix so-

lutions A and B in ratios other than the usual 1:1 ratio.

The amount of calcium and DNA in the mix can be kept

constant while the volume of solution B is varied from

0.7 to 1.2 times that of solution A. If a ratio of 1:1.2 is

used, then 110 ll of precipitate mixture per ml of culture

volume should be added to cells based on the standard

protocol (see below).

Fig. 3. Effect of phosphate concentration in the precipitation mixture

(DNA¼ 25 lg/ml) on turbidity, DNA binding, and expression of a

secreted protein (relative values for adherent CHO and adherent HEK

293 cells). The optimum fits with the minimal concentration of phos-

phate needed to precipitate most of the DNA (based on the centrifu-

gation assay). Higher concentrations of phosphate caused a too

abundant amount of larger particles with negative effects on trans-

fection efficiency.

2.2. Centrifugation assay

In the transfection cocktail, DNA is present at con-

centrations that allow easy detection by spectroscopy

(OD at 260 nm). To determine the level of DNA pre-

cipitation for a given set of conditions, solutions A and

B can be mixed with DNA, and the precipitate can be

removed by centrifugation for 30 s at 16,000g. The un-

precipitated DNA remaining in the supernatant can bequantified by measuring the OD at 260 nm using a mix

of solutions A and B as a blank. It should be verified

that the supernatant shows no absorption at 320 nm

since this indicates that not all the precipitate has been

removed by centrifugation or that further precipitation

occurred after centrifugation.

2.3. Turbidity test

The presence of a precipitate in the transfection

cocktail can be visually verified by checking if Tyndall

scattering occurs. Some experts in the field can visually

recognize precipitates that are highly efficient for

transfection. They describe such precipitates as ‘‘slightly

turbid’’ or ‘‘translucent’’ [17,40]. For a more objective

and accurate judgment, however, the turbidity4 of thecocktail can be measured at wavelengths of 320 nm or

higher [7,8,12]. The value 320 nm is the shortest wave-

length at which neither DNA nor any other components

of the precipitation mixture absorb. The turbidity

caused by the precipitate can be measured with a spec-

trophotometer in the absorption mode. As a quick and

efficient test, the turbidity assay is well suited to compare

batches of solutions, the mode of mixing, and otherparameters. The turbidity assay can be done with or

without DNA. In the absence of DNA, values of about

0.12 are expected (absolute values can depend on mix-

ing), but with DNA at a concentration of 25 lg/ml the

absorbance value is about 0.22. The effect of DNA on

turbidity is dose-dependent. This assay can be used to

compare different batches of DNA5.

For measuring turbidity, mix solutions A and B in thesame mode as for transfection (see Section 1.3), wait for

50 s, and then transfer the desired amount into a clean

cuvette. Since the turbidity is changing over time, the

OD at 320 nm is determined exactly 1min after mixing.

For a blank, either solution A or B can be replaced with

water.

2.4. Transfection of adherent cells

Transfection is most efficient when exponentially

growing cells are used. More precisely, it is the fraction

4 Turbidity is caused by small particles that scatter light.5 We have seen plasmid batches that have strong effects on

turbidity performing poorly in transfections.

M. Jordan, F. Wurm / Methods 33 (2004) 136–143 141

of the cell population in the late S phase of the cell cyclethat transfects well. These cells will undergo mitosis

shortly after the transfection to allow nuclear entry of

the DNA. In any case, cells should be transfected when

they are subconfluent in order to allow one more dou-

bling. Cells are typically seeded the day before the

transfection, but we have noticed that seeding CHO cells

2–4 h prior to transfection works as well. In this case, the

seeding density can be doubled.For the transfection of adherent cells in 12-well

plates, 1.5� 105 cells are seeded 16 h prior to transfec-

tion in 1ml DMEM/F12 medium containing 2% FCS.

The latter is necessary for cell attachment and for the

transfection step. The DNA is diluted into solution A.

Per milliter of cell culture medium 2.5 lg DNA6 is added

to 50 ll of solution A. Then 50 ll of solution B is added

and briefly mixed with the pipette. After 1min, 100 ll ofthe mix is added to each well. Cells are then incubated

for 4 h at 37 �C in 5% CO2. At the end of this period, the

medium is removed and the cells are treated for 1min

with 10% glycerol in PBS.7 After removal of the glyc-

erol, regular culture medium is added. Cells start to

express the protein within a few hours and achieve the

highest expression level about 1 day after transfection.

2.5. Transfection of suspended cells

The transfection of suspension cells has several ad-

vantages over the transfection of adherent cells: routine

passaging of the cells is easier, higher cell densities and

product titers can be achieved, and the method is scal-

able. We routinely grow HEK 293 cells in a serum-free

medium from JRH Bioscience and obtain maximum celldensities of 6 million cells/ml. The transfection, however,

is carried out in DMEM/F12 medium containing 1–2%

FCS [41].

For the transfection of HEK 293 cells in shaken 12-

well plates, our smallest cultivation system for suspen-

sion cells, exponentially growing cells are centrifuged

and resuspended at 5� 105 cells per ml in DMEM/F12

medium containing 2% FCS. Then, 1ml of cells is dis-tributed into each well immediately prior to transfec-

tion. For each transfection 2.5 lg of DNA is added to

50 ll solution A. Then, 50 ll solution B is added and

briefly mixed with the pipette. After 1min, 100 lltransfection cocktail is added per well. The plates are

shaken8 for 4 h at 37 �C under 5% CO2 [42] and then the

calcium is diluted by adding 1ml of growth medium.

The protocol can be directly applied to other suspension

6 Purified DNA is diluted to around 1mg/ml in TE (10mM Tris,

1mM EDTA, pH 7.4).7 As an optional step, cells are exposed for 30 s to 5mM EGTA in

PBS before the glycerol is added. This removes the precipitate and

reduces the risk of toxic side effects.8 Shaking diameter 20mm, rotation speed 180 rpm.

systems like spinner flasks or bioreactors [1,25,43]. Oursuccessful transfection within a 100L bioreactor proves

the scalability of the method and opens the door for

novel industrial applications of transient gene expres-

sion [44].

Most recently, we came up with an interesting mod-

ification of the transfection method for suspension cul-

tures. The DNA is simply diluted into solution A and

then added to the cells in the same medium and at thesame cell density as for the standard method to give final

concentrations of 12.5mM calcium and 5 lg DNA per

ml of medium. During the 4 h of transfection, the pH of

the medium has to be at 7.5 or higher, otherwise this

protocol does not work at all. All further steps are

identical to the standard method. This method is not

recommended for the transfection of adherent cells since

it has not yet worked in our hands.

3. Troubleshooting

3.1. Cytotoxicity

Calcium phosphate precipitates are cytotoxic in a

dose- and time-dependent manner. While soluble cal-cium ions rarely cause cell damage, calcium-containing

precipitates can be harmful to cells. Problems can get

worse when cells are exposed to an osmotic shock in

the presence of such precipitates. Toxic effects can be

avoided by limiting the exposure time to the precipitate

to a few hours. Once the precipitate is removed, sur-

viving cells recover quickly. In case of severe cytotox-

icity, the phosphate concentration of the medium(maximal 1mM) and the pH should be checked. In-

creasing the cell density during transfection and/or a

brief EGTA treatment at the end of transfection can

also help. In general, the presence of 2% serum protects

the cells from most of the negative effects of calcium

phosphate precipitates.

3.2. Poor expression

The transfection efficiency can be low if the cells are

not dividing or in a poor physiological state. In addi-

tion, transfectability of different cell lines varies within a

wide range. Transfection efficiency also depends on the

quality of the transfection solutions and plasmid DNA.

In some cases, a good transfection efficiency is not suf-

ficient. Transgene expression can be poor for a numberof other reasons that usually involve questions of the

toxicity or stability of the recombinant protein or the

strength of the transgene promoter. Thus, low expres-

sion is not always equal to a low transfection efficiency.

A useful approach is to systematically include a reporter

gene—representing only 2% or less of the total plasmid

DNA—to measure the transfection efficiency.

142 M. Jordan, F. Wurm / Methods 33 (2004) 136–143

3.3. Variability in transfection efficiencies

When doing transfections on different days, one has

to accept a certain level of variation. Significant varia-

tion can result if the cultivation of the cells is not done

under strictly reproducible conditions. By using the

same cells, DNA, transfection buffer, and medium for

repeated transfections we got consistent results. From

these results, we conclude that the formation of theprecipitate and its addition to the cells are not a major

source of variation. Twofold variations seen from 1

week to the next are acceptable to us.

4. Concluding remarks

Transfection of cells is a complex procedure that in-

cludes several steps that cannot be directly controlled.

While many parameters are known, it is not possible to

fully understand all of the molecular mechanisms in-volved in the process. Being aware of some key factors is

sufficient for dealing with such a complex system. Most

of the basic concepts are covered within this article and

more information can be obtained from the references.

The suggested protocols should help to get started

quickly. Rather than aiming for the highest efficiencies

possible, the given protocols should be reliable and work

at the first trial.We frequently compare the calcium phosphate tech-

nique with other methods available. Despite a rapidly

growing choice of efficient transfection reagents, this

method remains highly attractive. It is the only one that

involves small ions that are natural compounds of the

culture medium. Thus, no traces of synthetic chemical

components remain within the cells or the medium after

the transfection. This can be an advantage for the via-bility of cells and for the quality and purity of the

product. The authors are convinced that even 30 years

after the publication of the method, it remains a very

useful research tool to which improvements can still be

made.

Acknowledgment

The authors thank Dr. David Hacker for his helpful

comments on the manuscript.

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