GIBCO® Media Bottles - Green Fact Sheet 产品文献
What factors can contribute to rapid cell death/culture failure? 产品常问问题
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.
Why do I see cytotoxicity after performing transfection? Can you please help? 产品常问问题
Here (http://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-support/troubleshooting-transfection-experiments.html#2) are possible causes for reduced viability following transfection, along with suggested solutions. Please note that as per recent findings, antibiotics can be used in media during transfection. We have compared transfecting cells in medium with and without antibiotics in multiple cell lines, assessed both the transfection efficiency and toxicity, and found no difference. For stable transfections, wait at least 72 hours after transfection before adding selective antibiotics.
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.
How do you run a dose response curve for Geneticin (G418) or other selective antibiotic? 产品常问问题
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.