Cell Line Frequently Asked Questions

Cells are shipped in dry ice, and upon receipt should immediately be thawed for culture or stored in liquid nitrogen. Do not keep the cells in dry ice for an extended time. Do not use a -80°C freezer for long term storage. Cells can be stored in liquid nitrogen for many years. For very long-term storage, it is recommended to thaw cells after about 5 years, expand them again and freeze new vials.
Contact technical support at [email protected] if the cells are not frozen or in dry ice upon arrival.

Each vial of growing cells shipped from BPS Bioscience typically contains at least one million cells, unless specifically indicated on the website and the product datasheet. For some cells there may be slightly more than 2 million cells in a vial to ensure fast regrowth of the cell culture.
For growth arrested cells (used in our cell-based assay kits), the exact number of cells is usually not disclosed. Each vial provides a large number of cells that has been optimized for the specific cell-type and assay in consideration, so the assay can be performed immediately.

Yes, in addition to cell media and luciferase kits, we sell support reagents such as inhibitors and agonists.

The ONE-Step™ Luciferase Assay System consists of components A and B, which are mixed prior to adding the reagent to the cells. The reagent contains a lysis buffer, so there is no need to perform cell lysis separately. Just add the ONE-STEP™ reagent, wait for about 15 minutes, and read luminescence.

Growth arrested cells are engineered recombinant cell lines designed for single use. These cells are alive and suitable for one cell-based assay, but the cells cannot replicate and therefore do not proliferate in culture.

All cell lines are tested for bacterial and mycoplasma contamination prior to shipment. They are also tested for cell growth and viability at 24 and 48 hours after thawing. The corresponding Certificate of Analysis will be sent with the package containing the cells.

Sometimes things do not go as planned. Cell viability may be compromised due to improper shipping, storage, or thawing conditions. The 10% DMSO (Dimethyl Sulfoxide) contained in the freezing medium is toxic to cells and should be removed immediately upon thawing. Be sure to minimize the time cells are in the freezing medium when thawing. Please read the thawing protocol provided in the datasheet.
Optimal cell culture media and growth conditions are also specified in the datasheet and on our website. The thawing process is very stressful to the cells, so the culture medium used for thawing and during the first few days of culture should not contain selection antibiotics (such as geneticin, hygromycin or puromycin).
If cells thawed from the first vial do not grow as expected, contact us at [email protected] and we will troubleshoot with you before you thaw the second vial. Of note, it is recommended to expand the newly grown cells and freeze at least 10 vials at an early passage for future use.

Our recombinant cell lines have been engineered to express one or several genes following stable transfection of the cells. Briefly, cells are transfected with the gene of interest following standard procedures. A few days later, cells in which the gene of interest has integrated into the genome are selected using antibiotic pressure (geneticin, puromycin and hygromycin are the most common antibiotics used to generate our cell lines). Antibiotic pressure must be maintained during long term culture to avoid loss of the transfected gene.

Cell lines are guaranteed to be stable for up to 10 passages when grown in our media and using the appropriate selection antibiotics. In our hands they are stable for at least 20 passages most of the time, however we recommend starting over from an early-passage frozen vial at passage 15 to ensure reproducibility of results.

For best results, it is highly recommended to use optimized media from BPS Bioscience, which have been validated for each recombinant cell line. Other preparations or formulations of media may result in suboptimal performance. The composition of each thaw medium and growth medium is specified on our website. Keep in mind that the lot-to-lot variation and provenance of FBS (Fetal Bovine serum) may noticeably influence cell growth parameters.

To decrease packing and shipping costs for our customers, thaw and growth media or other components may be sent in the same package as the cells, in dry ice, and will be received frozen. This also ensures that media are received at the same time as the cells, as they are required for thawing and growing the cells. The media are stable when frozen and can be transferred to the recommended storage temperature (usually 4°C) without loss in activity.

A cell pool is obtained following antibiotic selection of a genetically modified cell population, without cloning. Thus, the cell pool contains all the cells that survived the antibiotic upon stable integration of the resistance gene. The resulting population may be somewhat heterogenous, however it better reflects the original cell population. Conversely, a cell line results from the cloning of antibiotic-selected cells using, for example, the limiting dilution method. Since a cell line originates from a single clone, it is homogeneous in terms of genetic modification or protein expression, however it may not reflect the original cell population as closely as a cell pool.

Cells that grow attached do not grow on regular plastic, therefore cells should always be plated on a cell culture-treated plate. It is preferable to use a clear-bottom plate to be able to visually observe the cells under a microscope after plating, before the experiment, and at the end of the experiment if necessary.

• Colorimetric assay: regular clear plastic cell culture plate.
• Luminescence assay: clear-bottom, white assay plate. White plates are used so that the light created by the luciferase in a well will be reflected up or down to the reader component and not be dispersed to neighboring wells through a clear wall.
• Fluorescence assay: clear-bottom, black assay plate. Black plates are used so that the fluorescence signal in a well will be reflected up or down to the plate to the reader component and not be dispersed to neighboring wells through a clear wall.

The optimal concentration of each antibiotic is initially determined for each cell line by performing a kill curve.

A kill-curve is a dose-response experiment in which cells are cultivated in the presence of increasing concentrations of a specific antibiotic for a period of one week to ten days. The minimum antibiotic concentration that is both required and sufficient to kill all the cells is then used for antibiotic selection, and for maintenance of the genetically modified cell line.

Protocol:

Plate the cells in a 24-well plate in their respective complete growth medium. Plate a number of cells that allows cells to reach approximately 30-50% confluency a day later. The day after plating, add increasing concentrations of the antibiotic of interest. Include a medium-only (no antibiotic) control. It is recommended to perform the dose-response in duplicates.
The useful range of concentration depends on the antibiotic.

For example:
G418                 0.1 to 2.0 mg/ml
Hygromycin   100 to 500 µg/ml
Puromycin     0.25 to 10 µg/ml

Replace the cell culture medium, maintaining the antibiotic concentration, every 3-4 days for up to 10 days. Examine the cells under a microscope every day for signs of cell death. Make note of the antibiotic concentration that is necessary and sufficient to kill all cells after 10 days.

The CO2 should be set at 5%.
CO2 present in the air (atmospheric) and dissolved in the cell culture medium affects the pH of the medium. This can be seen by changes in the color of media containing phenol red, which is a pH indicator: as pH decreases and the medium becomes more acidic, the phenol red turns orange, then yellow. Conversely, as pH increases and the medium becomes more basic, the phenol red turns pink.
Increasing the CO2 level decreases the pH of the medium and vice-versa. Atmospheric CO2 is regulated by the incubator and therefore remains constant. However, cell metabolism produces CO2, which is released in the medium as cells grow and decreases the pH of the medium. To stabilize the pH in the presence of higher levels of CO2, sodium bicarbonate NaHCO3 is used as a buffer. The concentration of sodium bicarbonate added to the medium is calculated to counteract specific amounts of CO2.
Media containing 1.5 to 2.2 g/L sodium bicarbonate are used to grow cells in 5% CO2. Media containing 3.7 g/L sodium bicarbonate are used to grow cells in 10% CO2. BPS Bioscience uses media containing the lower range of sodium bicarbonate, therefore cells grown in our media need to be maintained in a 5% CO2 incubator.

Trypan blue staining is routinely performed to verify the viability of cells in suspension. It is used for the colorimetric detection of dead cells.
Trypan blue is a highly charged molecule that does not pass through the cell plasma membrane. However, the dye will penetrate cells with a damaged plasma membrane. Therefore, damaged or dead cells appear blue while healthy cells exclude the dye and remain unstained. The method requires observation under the microscope with a manual count, or an automated cell counter with colorimetric detection capability.
Note that trypan blue staining is a very basic marker of cellular heath and does not provide mechanistic information on cell death. In addition, the dye quenches fluorescence and will interfere with fluorescence staining.