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Can I add antibiotics to OptiPRO SFM? Product FAQ

Answer

You can supplement OptiPRO SFM with 5mL/L Antibiotic-Antimycotic (Cat. No. 15240096)

Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.

Answer Id: E20494

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Matrix Invasion Assay Experiment Protocol

How do you run a dose response curve for Geneticin (G418) or other selective antibiotic? Product FAQ

Answer

The amount of antibiotic required to be present in culture media to select for resistant cells varies with a number of factors, including cell type. Good laboratory practice requires that the optimal concentration of Selective Antibiotic required to maintain and select cells must be determined for each set of growth conditions. Whenever experimental conditions are altered (including use of Selective Antibiotic from a different lot), the optimal concentration of the product should be re-evaluated.

Below is a brief protocol for performing a kill curve with Geneticin. Follow the general protocol for other antibiotics as well but use appropriate ranges for each antibiotic. For example, Geneticin: 100-1,500 ug/ml, Blasticidin: 1-10ug, Zeocin resistance gene: 100-1000 ug/ml.

Kill Curve Assay:

1. Dissolve Geneticin Selective Antibiotic in fully supplemented growth medium without antibiotics at a concentration of 5 mg/ml and filter using a 0.22 micron filter.
2. Prepare 6-well cell culture plates by adding Geneticin Selective Antibiotic to the growth medium to desired concentrations. A range from 100-1,200 µg/ml in 100 µg increments is recommended.
3. Treat cells with trypsin and dilute to a concentration of 4000 cells/ml.
4. Add 100 µl of cell suspension to each well and incubate plates in a humidified CO2 atmosphere at 37°C.
5. At 10 to 14 days, aspirate the supernatant and wash the cells with phosphate buffered saline and stain the cells with 0.5% methylene blue and 50% methanol for 20 minutes.
6. Score the plates by calculating percentage of survival by number of individual colonies for percent confluence.
7. Calculate the percentage of survival in the presence of each dilution of Geneticin Selective Antibiotic versus the percentage of survival in the absence of Geneticin.
8. Generate a dose response curve by plotting the percentage of survival on the y axis versus the concentration of Geneticin Selective Antibiotic in µg/ml on the x axis for both the sensitive and resistant cell lines.

If you are performing sequential transfections, one needs to establish dose response curve for the first antibiotic, create a stable and then perform a second does response curve on that stable in the presence of 2 antibiotics.

Answer Id: E4300

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Is CTS AIM-V Medium, without phenol red, without antibiotics ready-to-use? Product FAQ

Answer

Yes, CTS AIM-V Medium, without phenol red, without antibiotics is ready-to-use. Additional supplementation with cytokines and growth factors may be required for certain types of cells. Human serum or CTS Immune Cell SR (Cat. Nos. A2596101 and A2596102) can be added to AIM-V medium to promote cell expansion.

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Answer Id: E16474

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What is the difference between CTS AIM-V Medium and CTS AIM V SFM? Product FAQ

Answer

These two media have comparable performance and same cGMP quality. The main difference is that CTS AIM-V Medium (Cat. Nos. A3830801, A3830802, A4672701) does not contain phenol red and antibiotics. This medium formulation has been used as an ancillary reagent in a therapeutic cancer vaccine approved by FDA.

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Answer Id: E16475

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Why is the lot number on the Zeocin Selection Reagent (Cat. No. R25001) vial different from the one on the package? Product FAQ

Answer

Zeocin Selection Reagent (Cat. No. R25001) contains 1 g of Zeocin Selection Antibiotic supplied as a 100 mg/mL solution (Part No. 450430) in 8 x 1.25 mL vials. Part No. 450430 is associated with a specific lot number that is designated by our manufacturing tracking system, and this is the lot number that is listed on the package. The COA is also created based on this package part number and lot number.

Each of the 8 vials in turn has a Part No. 460509 that is associated with a different lot number designated by our manufacturing tracking system. This lot number is mainly used for our internal inventory purpose.

Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.

Answer Id: E17123

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How can I decontaminate my cultures? Product FAQ

Answer

When an irreplaceable culture becomes contaminated, researchers may attempt to eliminate or control the contamination.

1. Determine if the contamination is bacteria, fungus, mycoplasma, or yeast. Read more here to view characteristics of each contaminant.
2. Isolate the contaminated culture from other cell lines.
3. Clean incubators and laminar flow hoods with a laboratory disinfectant, and check HEPA filters.
4. Antibiotics and antimycotics at high concentrations can be toxic to some cell lines. Therefore, perform a dose-response test to determine the level at which an antibiotic or antimycotic becomes toxic. This is particularly important when using an antimycotic such as Gibco Fungizone reagent or an antibiotic such as tylosin.

The following is a suggested procedure for determining toxicity levels and decontaminating cultures:

1. Dissociate, count, and dilute the cells in antibiotic-free media. Dilute the cells to the concentration used for regular cell passage.
2. Dispense the cell suspension into a multiwell culture plate or several small flasks. Add the antibiotic of choice to each well in a range of concentrations. For example, we suggest the following concentrations for Gibco Fungizone reagent: 0.25, 0.50, 1.0, 2.0, 4.0, and 8.0 µg/mL.
3. Observe the cells daily for signs of toxicity such as sloughing, appearance of vacuoles, decrease in confluency, and rounding.
4. When the toxic antibiotic level has been determined, culture the cells for two to three passages using the antibiotic at a concentration one- to two-fold lower than the toxic concentration.
5. Culture the cells for one passage in antibiotic-free media.
6. Repeat step 4.
7. Culture the cells in antibiotic-free medium for four to six passages to determine if the contamination has been eliminated.

Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.

Answer Id: E11916

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Will improper storage of my tissue culture reagents affect the growth rate of my culture? Product FAQ

Answer

Yes. Store animal sera at -5 to -20 degrees C. Store media at 2 to 8 degrees C; use within recommended shelf life period. Store complete media (supplemented) at 2 to 8 degrees C, and for complete medium the recommended shelf life is 2 to 4 weeks. Additionally, minimize exposure of sera and media to light.

Answer Id: E11825

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What factors can contribute to rapid cell death/culture failure? Product FAQ

Answer

There are a number of events that can contribute to this:

1. Incorrect CO2 levels-monitor the level of CO2 manually with a Fyrite kit, available from Bacharach (http://www.bacharach-inc.com/fyrite_analyzers.htm). Check if the manual readings concur with the readings displayed on the incubator. If the incubator has a trace readout, check the printout for fluctuations in CO2 level. Check the settings to insure that CO2 levels are set at appropriate levels for your cell line (usually between 5 and 10%). Check line connections frequently for leaks. Avoid frequent opening and closing of incubator doors.
2. Temperature fluctuations in the incubator-Monitor the temperature of incubator with a good thermometer inside the incubator.
3. Fungizone or other preventive antibiotics/antimycotics are present at toxic concentrations-use at recommended levels.
4. Humidity is incorrect-check the water level in the water pan. Humidity is vital to appropriate gas exchange for many types of cells and media (i.e., appropriate CO2 levels are largely irrelevant for most cultures if the humidity level is not high enough).
5. Incorrect osmotic pressure in medium-check osmolality of complete medium. Most mammalian cells can tolerate an osmolality of 260 to 350 mOsm/kg. Additions of reagents such as HEPES and drugs may affect osmolality.
6. Contamination by microorganisms-bacterial and fungal contaminations are usually easily visible; symptoms of mycoplasma contamination are more subtle, and careful monitoring of culture morphology and regular testing are necessary to detect this type of contamination.
7. Inappropriate medium is being used-double-check that the medium used is appropriate for your cell type and culture application. For example, ensure that the medium being used for serum-free culture is actually designed for serum-free culture; make sure that appropriate selective drugs are used at appropriate levels; check the expiration dates for the reagents being used; and store medium at appropriate temperatures in the dark.

Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.

Answer Id: E11905

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I performed an LR reaction, followed by transfection into Sf9/Sf21 insect cells, but do not see any signs of infection even though it's been 72 hours. What should i do? Product FAQ

Answer

Please see our recommendations below:

- Check the LR reaction by PCR analysis prior to transfection into insect cells.
- We recommend using Grace's Insect Cell Culture Medium, Unsupplemented during the transfection experiment instead of serum-free medium, as components in serum-free medium may interfere with transfection.
- Ensure that FBS, supplements, or antibiotics are not included during transfection, as the proteins in these materials can interfere with the Cellfectin II Reagent.
- Use the LR recombination reaction using the pENTR/CAT plasmid as a positive control and Cellfectin II Reagent only (mock transfection) as a negative control.
- Ensure that cells are in the log phase of growth with >95% viability, and the amount of cells are in accordance with the suggestions in the manual.
- Cells may not show signs of viral infection for up to a week depending on transfection efficiency; continue culturing and monitor cells daily for signs of infection.

Answer Id: E9459

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What is the recommended method to seed PSC to start Definitive Endoderm Induction? Product FAQ

Answer

When seeding PSCs for Definitive Endoderm induction, cells should be plated as very small clumps using Accutase Reagent. They can also be seeded as singularized cells using TrypLE Reagent. To promote cell survival, you can treat the cells overnight with ROCK inhibitors including RevitaCell Supplement , Y27632, or Thiazovivin.

Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.

Answer Id: E9384

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What is the PSC Cryopreservation Kit? Product FAQ

Answer

The PSC cryopreservation kit contains xeno-free PSC Cryopreservation Medium, which is a ready-to-use solution for the cryopreservation of early passage pluripotent stem cells (PSCs), and Gibco Revitacell Supplement (100X), a chemically defined recovery supplement for use in the post-thaw culture medium. When used in combination, these reagents help minimize loss of cell viability, maximize post-thaw recovery, and minimize unwanted differentiation of PSCs. This kit can also be used to cryopreserve and recover peripheral blood mononuclear cells (PBMCs) to improve post-thaw cell viability and recovery.

Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.

Answer Id: E12461

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Does Efficient-Pro Medium require supplementation for use? Product FAQ

Answer

When using a CHO cell line with a selection system other than the glutamine synthetase (GS) system, we recommend adding 2-8 mM L-glutamine or GlutaMAX Supplement to Efficient-Pro Medium. Additionally, we recommend making sure that glucose levels do not fall below 2 g/L in culture by supplementing with glucose as needed. Also, if clumping becomes an issue, then add anti-clumping agent (ACA) to your cultures at 1% v/v.

Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.

Answer Id: E20376

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Does Efficient-Pro AGT Medium require supplementation for use? Product FAQ

Answer

When using a CHO cell line with a selection system other than the glutamine synthetase (GS) system, we recommend adding 2-8 mM L-glutamine or GlutaMAX Supplement to Efficient-Pro AGT Medium. Additionally, we recommend making sure that glucose levels do not fall below 2 g/L in culture by supplementing with glucose as needed. Also, if clumping becomes an issue, then add anti-clumping agent (ACA) to your cultures at 1% v/v.

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Answer Id: E20381

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Does High-Intensity Perfusion (HIP) CHO Medium require supplementation for use? Product FAQ

Answer

When using a CHO cell line with a selection system other than the glutamine synthetase (GS) system, we recommend adding 2-8 mM L-Glutamine or GlutaMAX Supplement to High-Intensity Perfusion (HIP) CHO Medium. Additionally, we recommend making sure that glucose levels do not fall below 2 g/L in culture by supplementing with glucose as needed. Also, if clumping becomes an issue, then anti-clumping agent (ACA) can be used.

Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.

Answer Id: E19144

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