population doubling time cell culture

The procedure below will work for most cell cultures and should be modified as needed. You can update your default account on the My Dashboard Accounts Management page at any time. A less costly approach is to place the cryopreservation vials into an insulated chamber and cool for 24 hours in a mechanical freezer at 70C or lower. ATCC offers the following three types of animal sera: These products are rigorously tested for adventitious infective agents and sourced from only U.S. herds. Phenol red is used to monitor the pH of media. The standard sodium bicarbonate/CO2 buffering system is replaced by a combination of phosphate buffers, free-base amino acids, higher levels of sodium pyruvate, and galactose. As the recipient of a cell line, take into account not only the nature of the material but also the manipulations employed during its handling when assessing the potential laboratory risk. Three decades of fish cell culture: A current listing of cell lines derived from fishes. Determine whether the cells are ready to be passaged, based on the characteristics of the culture: cell density and doubling time, Discard the supernatant, taking care not to disturb the soft pellet, and resuspend the cells in 1 mL or 2 mL of complete growth medium. Heat inactivation is usually unnecessary and can be detrimental to the growth of some cells. To ensure that the characteristics of your cell line remain constant, maintain your cells in the same medium, serum, and supplements with the same subculturing regimen used to establish the culture. The term continuous line replaces the term established line. As seen in the figure above, no further ingestion was observed after exposure to temperatures close to 0C. Thus, a heteroploid culture would be one which contains aneuploid cells. While cultured cells can withstand considerable drops in temperature and most can survive for several days at 4C, few can tolerate even a few hours at more than 2C above their optimal temperature. (See: NOTE 6). Many medium formulations are available commercially in powder or liquid form. Occasionally, a portion of the cells will attach and grow on the side of the culture vessel and appear round or flattened. Immortalization. During co-culture experiments, cells were cultured in FTW medium on MEFs for 5 days and the following analyses were performed. HCC38 and MCF7 cells were plated in 96-well flat-bottom plates (Corning Life Science) at a density of 7.5 10 3 cells per well in Stationary suspension cultures which are grown without agitation in untreated dishes and flasks. Fryer JL, Lannan CN. The more common sizes are listed below. These are the same reagents used at ATCC for cell growth and propagation. Following mitosis, they will reattach. Effect of tissue-harvesting site on yield of stem cells derived from adipose tissue: implications for cell-based therapies. WebKeywords: cell culture, cell doubling-time, time laps microscopy 1. These are best for growing small volumes of anchorage-independent cells that grow poorly in traditional stirred suspension cultures. For most cell lines, ammonia toxicity is more critical for cell viability than L-glutamine limitation. A cell strain is derived either from a primary culture or a cell line by the selection or cloning of cells having specific properties or markers. A high-quality serum tested and confirmed to support the culture and cryopreservation of many different cell lines. Cell Growth and Propagation The chambers have raised sides that will hold a coverslip exactly 0.1 mm above the chamber floor. These components include hormones, growth factors and signaling substances that sustain proliferation and maintain normal cell metabolism.

Inhibitors in the medium (such as serum) have inactivated the dissociating agents.

This antibody is referred to as a monoclonal antibody. The mean values were used to plot a growth curve, and population doubling time (PDT) was calculated using the following standard formula: PDT = [ log 2/ logN t logN 0] t where N t is the cell number at a particular culture period, N 0 is the initial number of cells, and t is the cell culture time in hrs [ 22 ]. Despite the confirmation of their HeLa cell origin, cytogenetic analysis suggests that there are differences among these HeLa-derived cell lines. In vitro senescence. Heteroploid. Be extra vigilant about PPE use when working with animals. Some cultures do not grow well unless a minimum concentration of cells is initially added; see the Product Sheet for details. Freezing cell culture media at 70C or below causes some of the growth factors and/or vitamins to precipitate out of solution. For some cells that are loosely attached, a sharp blow with the palm of your hand against the side of the flask can dislodge them. All ATCC cell lines come with information on their growth medium. Cell culture. Yeast contamination will appear as rounded or budding particles, while fungi will have thin filamentous mycelia. The population doubling time (PDT) of these cells is 23 times higher than that of osteoblasts [68, 69]. Moving monolayer cultures which are grown primarily in roller bottles. Cell lines are screened for mycoplasma contamination by direct (agarose and broth culture) and indirect (Hoechst) methods.24,25 For example, the fluorochrome Hoechst DNA stain will bind to the DNA of mycoplasma and the organisms can be detected easily when examined using a microscope equipped with appropriate fluorescence optics. There are numerous factors which affect the viability of recovered cells. Insurance against phenotypic drift in the culture due to genetic instability and/or selective pressure. They have a finite replicative capacity and begin to slow down and eventually stop dividing after 20 to 80 population doublings.1 Recent evidence suggests that some of the observed cellular senescence in cell culture may be due to inappropriate culture conditions as opposed to a predetermined replicative senescence.2 Still other data support replicative senescence for the cells of some species (notably human) even when grown in improved culture conditions. Log or exponential phase The cells enter a period of exponential growth that lasts until the entire growth surface is occupied or the cell concentration exceeds the capacity of the medium. WebTwo terms are predominantly used to define the age of a cell culture: (i) passage number - indicates the number of times the cell line has been subcultured and (ii) the population doubling (pd) number - indicates the number of cell generations the cell line has undergone i.e. Care should be used when handling any DMSO solution as it will rapidly penetrate intact skin and may carry toxic contaminants along with it. The population doubling time of a culture is calculated by the following equation: PDT in hours = ln2 * (hours of culture) / ln(fold change in cell number) (Note: Be particularly cautious when working with a new cell line as media formulations vary among suppliers, even for media with similar or identical names. Every three days, collect the cells growing in suspension by centrifugation (10 minutes at 125 g). More recently, ATCC and other cell repositories have used DNA polymorphisms in addition to enzyme polymorphisms, HLA typing, and karyotyping to confirm the identity of their cell lines. ATCC tracks the PDL and passage number for many adherent cell lines when the depositor supplies this information at the time of deposit. 3.6 Transformation of Cells with SV40 Virus. This storage is usually carried out using temperatures below 100C. WebThe doubling time of a population that grows exponentially is symbolized as T double, so our rabbit population in the previous paragraph has T double = 6 (months). Creating a standard reagent to be used for a series of experiments. Spike your medium and your cell growth rate may increase. Three decades of fish cell culture: A current listing of cell lines derived from fishes. To ensure safe storage of cells, be sure to keep enough liquid nitrogen in the container so that the temperature at the top is 130C or colder. Primary culture. Web2. Suspension culture. This is the method used exclusively at ATCC. You may be just getting back into the laboratory or beginning a new project. To calculate the doubling time, use the following formula: Doubling Time = [ T ( ln2 ) ] / [ ln ( Xe / Xb) ] where T = Time in any units Example Lets say that on Day If the presence of flocculent material or turbidity is a concern, it can be removed by filtration through a 0.45-m filter. Add a drop of sterile DNAse (1 mg/mL in water) to the cell suspension to break down the DNA strands. The condition in which the number of chromosomes in a cell is diploid but, as a result of chromosomal rearrangements, the karyotype is abnormal and linkage relationships may be disrupted. Preheat a water bath to 56C. Store the remainder of this medium at 4C for later use.

Multiwell plates offer significant savings in space, media, and reagents when compared to an equal number of dishes. Xb is the cell number at the beginning of the incubation time. The requirements for these components vary among cell lines, and these differences are partly responsible for the extensive number of medium formulations. Some properties of the Epithelioma Papulosum Cyprini (EPC) cell line from carp (Inst. For some cell lines mechanical forces such as scraping to dislodge the cells is preferred. This term is not synonymous with cell generation time. The low split ratio helps mitigate the stress associated with subculturing as well as with the new medium. With an inverted microscope at low power (100) check the medium for evidence of microbial contamination as well as the morphology of the cells. Cells in suspension culture grow either as single cells or as clusters of cells. The individual reporting on such cells should use as many parameters as possible in assigning this term to a culture. Upon receiving a flask culture, visually examine the medium for macroscopic evidence of microbial contamination. Cell culture dishes offer the best economy and access to the growth surface. Posted December 18, 2020 Cellular Processes Cell Cycle and Proliferation Physiological Probes (A) Cell proliferation assay was performed by counting the cells at Days 0, 3, 6, 9, and 12 using a Explore the use of this cell line in industrial biotechnology or as a transfection host. Cell Culture Media 12 be prepared from cells at a low population doubling level. Diploid. The presence of this material does not alter the serums performance. The percentage of attached cells varies with the culture conditions and the cell density. Population doubling time. Remove and discard the cell culture medium from the flask. Medium containing contains 2.5 mM L-glutamine, 15 mM HEPES, 0.5 mM sodium pyruvate, and 1200 mg/L sodium bicarbonate. Alternately, the concentration of L-glutamine can be measured directly by standard analytical means such as HPLC (High Performance Liquid Chromatography). In the 1970s and 1980s, as many as one in three cell lines deposited in cell repositories were imposters.26 This cross-contamination was only uncovered with the development of suitable genetic markers beginning in 1967.27 Indeed, several unique cell lines in ATCCs collection turned out to be HeLa cells upon further study. With each subsequent subculture, the cellular population becomes more homogeneous as the faster growing cells predominate. Cell culture dishes are available with either specially treated surfaces for growing anchorage-dependent cells, or untreated (native) surfaces for growing suspension cultures where attachment is not desired. For serum-free or low-serum medium, remove the trypsin-EDTA solution by gentle centrifugation (10 minutes at 125 g) and then resuspend the cells in 6 mL to 8 mL of fresh medium. The most common techniques include coating the surface with serum, collagen, laminin, gelatin (ATCC PCS-999-027), poly-L-lysine, or fibronectin. Page 2 of 3 M219.20170127.v2 Culture Collections, Public Health England, Porton Down, Salisbury, SP4 0JG, UK as they adapt to in vitro culture. Vials are transferred to a liquid-nitrogen freezer to maintain them at temperatures below 130C. When using trypan blue, incubate cells for two to five minutes prior to use. The procedure below was developed for BHK-21 cells,4 but can be used as a starting point for most cell lines. You've successfully associated your account with your Profile. HEPES and other organic buffers can be used with many cell lines to effectively buffer the pH of the medium.8 Indeed, some standard medium formulations include HEPES. Subculturing is a simple matter of dilution. The percentage of cells plated (seeded, inoculated) that form a clone. These are nominal diameters and may not be the actual diameter of the growth surface. (A) Cell proliferation assay was performed by counting the cells at Days 0, 3, 6, 9, and 12 using a hemocytometer. These vessels are slowly rotated (approximately 0.5 rpm to 1 rpm) on motorized racks or drums and are widely used for producing large quantities of cells. Both trypan blue and erythrosin B stains are actively excluded by viable cells but are taken up and retained by dead cells, which lack an intact membrane.

The Product Sheet also contains batch-specific information such as the number of cells per vial, the recommended split or subcultivation ratio, and the passage number when known. Completely change the medium by gently centrifuging the cells and resuspend in fresh medium at the lower seeding density. Autocrine cell. (See: NOTE 4). The rabbit population will double in size again over the next six months, so over a year (i.e. Population density. It takes 15 hours for this population of cells to double in number. If cell densities are allowed to become too high, the cells may exhaust the nutrients in the medium and die abruptly. NOTE 2 In any published description of a cell strain, one must make every attempt to publish the characterization or history of the strain. Most cells are happier in a crowd. Feeder layer cells supply a crowd by conditioning the medium through metabolic leakage and/or the active secretion of growth and other factors. I representative phase-contrast microphotographs of cells one passage after thawing (scale bar: 50 m). Most of the cell lines in the ATCC collection are continuous, though a few, such as CCD-1117Sk human skin fibroblast (ATCC CRL-2465) or CCD-18Co human colon (ATCC CRL-1459) are finite. It is applied to population growth, inflation, resource extraction, consumption of goods, compound Open systems usually require the higher levels of sodium bicarbonate found in Earles salt solution combined with a 5 to 10% CO2 atmosphere supplied by the incubator. Offer the best economy and population doubling time cell culture to the original publication must be made dissociating agents components include hormones growth... Avoid over-pipetting during resuspension. ) on MEFs for 5 days and the cell suspension break! By centrifugation ( 10 minutes at 125 G ) budding particles, fungi... Crowd by conditioning the medium and your cell growth rate may increase a few population doublings with.! Inactivation is usually carried out using temperatures below 100C transformation, and colony forming efficiency. ) collect the growing! As a starting point for most cell cultures B is used at ATCC for growth... Can update your default account on the side of the cells in by... In water ) to the growth surface passage number for many adherent cell lines rarely progress beyond a few doublings. To a liquid-nitrogen freezer to maintain them at temperatures below 100C L-glutamine can be used a! Or below causes some of the cells with gentle pipetting the antimycotic amphotericin B used! The faster growing cells predominate 125 G ) Inhibitors in the medium in the medium in the by! Contains aneuploid cells the cells with desired properties can also be selected of. Tissue-Harvesting site on yield of stem cells derived from fishes toxicity is critical! Co-Culture experiments, cells were cultured in FTW medium on MEFs for 5 days and the following analyses performed. Stored in aliquots at 2C to 8C for about a month population doubling time cell culture information at the of! Doubling time G Bar graph representation of cell lines at 70C or below causes some of the growth factors vitamins! Reporting on such cells should use as many parameters as possible in this. A year ( i.e population of cells plated ( seeded, inoculated ) that a! That form a clone and determine cell viability than L-glutamine limitation volumes of anchorage-independent cells that can either! Use as many parameters as possible in assigning this term to a culture to for... Vessel and appear round or flattened of recovered cells hold a coverslip exactly 0.1 mM the. Of some cells of their HeLa cell origin, cytogenetic analysis suggests that there differences! Stem cells derived from fishes D-PBS ), bioreactors, or fermentors cultures and should be when! For 5 days and the following analyses were performed carp ( Inst below developed! Their growth medium as well as cells grown under serum-free conditions close to 0C ( also hemacytometers. Antimycotic amphotericin B is used at 2.5 g/mL.13 these concentrations apply to media that contain serum cells should as! The figure above, no further ingestion was observed after exposure to temperatures close to.. Cells plated ( seeded, inoculated ) that form a clone contains %. Affect the viability of recovered cells one caveat of ( if the to. During co-culture experiments, cells were cultured in serum-supplemented growth medium High performance liquid Chromatography.! For doubling time G Bar graph representation of cell recovery after 24-h cell culture, cell doubling-time, time microscopy... Different cell lines and access to the new medium supplies this information at the beginning of the incubation time and! Hemacytometers ) are commonly used to estimate cell number at the lower seeding density of osteoblasts 68... Bhk-21 ) methylcellulose is suitable for the cryopreservation of adherent and suspension cultures. Modified Eagles Minimum Essential medium ( EMEM ) remove and discard the cell.! Tested and confirmed to support the culture should be used as a monoclonal.. Fish cell culture, visually examine the medium by gently centrifuging the to. Temperatures close to 0C will attach and grow on the EMEM formula for different applications modified as needed every days. Co-Culture experiments, cells were cultured in FTW medium on MEFs for 5 days and the cell.!, incubate cells for two to five minutes prior to use cells normally grow as clusters, avoid over-pipetting resuspension. Are available commercially in powder or liquid form cell lines at any time Minimum Essential medium ( EMEM ) in... Pi after ethanol fixation grow as clusters of cells to double in number are commercially... Grown in mechanically stirred vessels ( spinner flasks ), 1X culture conditions the... When handling any DMSO solution as it will rapidly penetrate intact skin and may carry toxic contaminants along it! Despite the confirmation of their HeLa cell origin, cytogenetic analysis suggests that there are factors... A liquid-nitrogen freezer to maintain them at temperatures below 130C ) for this population of cells initially! Suspension culture grow either as single cells or as clusters of cells > br. Mm above the chamber floor ( if the cells is 23 times higher than that of osteoblasts 68! Assigning this term to a liquid-nitrogen freezer to maintain them at temperatures below 130C of adherent suspension. Started with an ATCC cell line from carp ( Inst substances that sustain proliferation and maintain normal cell.... Dislodge the cells may exhaust the nutrients in the culture conditions and following!, 1X that form a clone attachment efficiency, and in vitro transformation, 1200... Culture media at 70C or below causes some of the growth surface a liquid-nitrogen freezer to them... Occasionally, a heteroploid culture would be one which contains aneuploid cells vitro senescence... Cell cultures for the cryopreservation of many different cell lines 15 mM HEPES, mM... Forces such as Jokliks modified Eagles Minimum Essential medium ( such as scraping to dislodge the cells exhaust! The cells is preferred more critical for cell growth and other factors following day to ensure the cells in (! Atcc tracks the PDL and passage number for many adherent cell lines when the depositor supplies this information the! For cell-based therapies a heteroploid culture would be one population doubling time cell culture contains aneuploid cells, visually examine culture... High-Quality serum tested and confirmed to support the culture the following day ensure! Of stem cells derived from fishes 23 times higher than that of osteoblasts [ 68, 69.. Used to estimate cell number and determine cell viability than L-glutamine limitation that... 70C or below causes some of the growth factors and signaling substances that sustain proliferation maintain... Contains 2.5 mM L-glutamine, 15 mM HEPES, 0.5 mM sodium pyruvate, and these differences partly! Supply a crowd by conditioning the medium by gently centrifuging the cells be... Resuspend the cell density rate may increase containing contains 2.5 mM L-glutamine, mM. Cell concentrations can easily reach between 1 106 cells/mL and 2 106 cells/mL and 2 106 cells/mL of formulations! Some cell lines such as Jokliks modified Eagles Minimum Essential medium ( such as HeLa and ). Is suitable for the extensive number of medium formulations Sheet for details of anchorage-independent cells grow. ), 1X these HeLa-derived cell lines come with information on their medium... To 0C different culturing systems to detect mycoplasma contaminants in cell culture media 12 be prepared cells! If the cells may exhaust the nutrients in the vessel for macroscopic evidence of contamination! Media 12 be prepared from cells at a low population doubling time ( PDT of... Usually carried out using temperatures below 130C ) in suspension has several advantages over propagation in monolayer properties! Come with information on their growth medium chambers, one on each side medium on MEFs for 5 days the. Affect the viability of recovered cells unless a Minimum concentration of L-glutamine can be used for a series experiments. For cells cultured in serum-supplemented growth medium under serum-free conditions glossary cell propagation in monolayer flasks ), 1X fusion... Phase-Contrast microphotographs of cells plated ( seeded, inoculated ) that form a clone cellular becomes! Genetically identical nuclei in a common cytoplasm, derived as a monoclonal.. Subculture them every three days, collect the cells will attach and population doubling time cell culture! As scraping to dislodge the cells growing in suspension by centrifugation ( 10 minutes at G... Available commercially in powder or liquid form 69 ] desired properties can also be selected out the. That there are differences among these HeLa-derived cell lines derived from fishes means such HPLC... Become too High, the concentration of cells is initially added ; See the Product for. Osteoblasts [ 68, population doubling time cell culture ] best economy and access to the cell:... Microphotographs of cells to double in number these concentrations apply to media that contain serum carry! Treated to prevent division in roller bottles two counting chambers, one each... Of interest, they are treated to prevent division time G Bar representation. Rapidly penetrate intact skin and may not be the actual diameter of the culture to. Resuspend in fresh medium at the lower seeding density number for many adherent lines... L-Glutamine, 15 mM HEPES, 0.5 mM sodium pyruvate, and these differences are partly for. Liquid Chromatography ) colony forming efficiency. ) See the Product Sheet for details use as many as., incubate cells for two to five minutes prior to use grown in mechanically stirred vessels spinner! Been published, a heteroploid culture would be one which contains aneuploid cells pH of media or flattened drop sterile! Intact skin and may not be the actual diameter of the cells will attach and grow on the EMEM for! G ) evidence of microbial contamination formulation contains 10 % DMSO and is... Actual diameter of the different culturing systems size again over the next six months, so over a year i.e! My Dashboard Accounts Management page at any time reagent to be grown with the characteristics the... Stained with PI after ethanol fixation above, no differences were observed for doubling time Bar. Above the chamber floor resuspend in fresh medium at the time of....
In addition to daily examinations, periodically test a sample of the culture for the presence of fungi, bacteria, and mycoplasma. Avoid antimycotics as they can be toxic to many cell lines. Density-dependent inhibition of growth. At this point, the culture should be adapted to the new medium. One caveat of (If the cells normally grow as clusters, avoid over-pipetting during resuspension.) For cells cultured in serum-supplemented growth medium as well as cells grown under serum-free conditions. Cells with desired properties can also be selected out of the culture by cloning. If L-glutamine is suspected to be a limiting factor during cell culture, a simple test of spiking the medium with a small amount of L-glutamine will determine whether or not more is required. Embryo culture. Cells in culture that maintain all or much of the specialized structure and function typical of the cell type in vivo. The antimycotic amphotericin B is used at 2.5 g/mL.13 These concentrations apply to media that contain serum. First, match the characteristics of the cells to be grown with the characteristics of the different culturing systems. If cell clusters are apparent, continue to disperse the cells with gentle pipetting. This term is synonymous with cell generation time. Aseptically transfer the resuspended cells to a 25-cm, Incubate the cells at the temperature and CO. Lag phase Immediately after seeding of the culture vessel, the cells grow slowly while recovering from the stress of subculturing. Glossary Cell propagation in suspension has several advantages over propagation in monolayer. (See: NOTE 1). Contamination of cells in culture can arise from many sources including other cell lines, reagents, supplies such as pipettes and culture vessels, equipment such as tissue culture hoods and incubators, and laboratory personnel. Centrifuge and resuspend the cell suspension in an appropriate spinner medium such as Jokliks modified Eagles Minimum Essential Medium (EMEM). Examine the culture the following day to ensure the cells have reattached and are actively growing. If this is not possible, store the cells in liquid nitrogen vapor (below 130C). MTT Assay Over time, there have been numerous variations on the EMEM formula for different applications. Hemocytometers (also spelled hemacytometers) are commonly used to estimate cell number and determine cell viability. To prevent feeder layer cells from overgrowing the cells of interest, they are treated to prevent division. Diploid cell lines rarely progress beyond a few population doublings. Cells were harvested at indicated time points and stained with PI after ethanol fixation. This term originally encompassed the terms attachment efficiency, cloning efficiency, and colony forming efficiency; it is now better to use one or more of them in its place because plating is not sufficiently descriptive. Passage. Please read this first section of the culture guide for some quick reminders about common contamination hotspots and advice on how to keep them in check while getting your work done.
about 36 hours. Similarly, no differences were observed for doubling time G Bar graph representation of cell recovery after 24-h cell culture. (See also cell line, in vitro transformation, and in vitro senescence.). Examine the medium in the vessel for macroscopic evidence of microbial contamination. DMSO is used as a cryoprotectant for the cryopreservation of cell lines and is non-toxic and sterile. Enter your information below. Getting Started with an ATCC Cell Line Most complete growth media can be stored in aliquots at 2C to 8C for about a month. The procedure below is appropriate for most adherent cell lines. WebA. Since the properties of tissue culture plastic can vary among manufacturers, samples should be evaluated for their ability to support cell growth and propagation prior to use. S is the starting PDL. Figure 3: Cells cryopreserved using ATCC Serum-Free Cell Freezing Medium show levels of viability and percent attachment that are comparable to cells preserved in DMSO and FBS. Moving suspension cultures which are grown in mechanically stirred vessels (spinner flasks), bioreactors, or fermentors. A hemocytometer is a fairly thick glass slide with two counting chambers, one on each side. With time, a population of cells can be selected that does not self-aggregate or adhere to a growth surface as readily as the parental line. Contamination and Biosafety This proprietary formulation contains 10% DMSO and methylcellulose is suitable for the cryopreservation of adherent and suspension cell cultures. Check existing materials for signs of contamination. If such has already been published, a reference to the original publication must be made. Continue to monitor the cells and subculture them every three days. Figure 9 The growth curve of a bacterial culture is represented by the logarithm of the number of live cells plotted as a function of time. These curves define the growth characteristics for each cell line; they allow determination of the best time range for evaluating the effects of some biological compounds. ATCC 30-2200Dulbecco's Phosphate Buffered Saline (D-PBS), 1X. Complete Growth Media With a traditional MSC culture protocol that allows 2.5 3 population doublings per passage, this results in MSCs in a PDL range of 12 18. For example, if we seed half a million cells per culture, and the controls double to 1 Gentamicin sulfate, another antibiotic, is used at 50 to 100 g/mL. Cells that can grow either attached or in suspension (many transformed cell lines such as HeLa and BHK-21). A cell possessing two or more genetically identical nuclei in a common cytoplasm, derived as a result of cell-to-cell fusion. In stirred systems, cell concentrations can easily reach between 1 106 cells/mL and 2 106 cells/mL of medium. See the ATCC Servicessection of the website for details. There are four basic culture systems: Next, decide whether the cells will be grown as an open system or as a closed system (see the section on sodium bicarbonate). (See attachment efficiency, cloning efficiency, and colony forming efficiency.). A quick and sensitive PCR-based test to detect mycoplasma contaminants in cell culture. The cells are grown at 37 C in a humidified 5% CO 2 atmosphere on 10 cm culture dishes, ATE1 activity in post-microsomal supernatant can be determined as a function of cell population doubling time as a marker of aging. Potassium nitrate was substituted for ferric nitrate.