Troubleshooting
1. Recovery from proliferation failure of culturing human lymphocytic cells
Proliferation failure and/or cell death are often observed after thawing especially in human lymphocytic cell lines. This is possibly because these cells require autocrine factor(s) for their survival and growth. Please try the following procedures.- It is important to remove DMSO contained in the cryopreservation medium. Suspend the vial contents in 10 ml of culture medium and centrifuge it once at 1000 rpm for 3 min. Avoid excessive dilution of the first culture. Re-suspend the cells in 5 ml of culture medium and start cultivation. To achieve a high density of cells, it is helpful to tilt the flask in a CO2 incubator for several days.
- Increase serum concentration in culture medium to 20%. The autocrine factor(s) may be substituted with serum.
- RPMI1640 medium is often used for human leukemic cell lines, however, the pH of this medium can increase towards alkaline, which impedes growth. If the pH of the culture medium when in the CO2 incubator is too high, please adjust it.
2. Contact information for technical inquiries
If you have a problem culturing cells, first of all, check Q&A for troubleshooting. JCRB accepts complaints within 3 months after shipment. Please make inquiries using web contact form, and describe your culturing procedure as following:- Your Order Number (F1xxxxxx, A1xxxxxx or E1xxxxxx), cell number, cell name and lot number
- How did you preserve the frozen vial? In liquid nitrogen, or -80°C freezer? How long did you store the cells?
- Did you centrifuge the cell suspension to remove DMSO?
- Media used: manufacturer and catalog number.
- Did you use heat-inactivated serum or not?
- Additives detail and concentration (such as serum, L-glutamine, antibiotics).
- Scale (size) and number of flasks / dishes at the initiation of culture.
- Flasks and dish: manufacturer and catalog number.
- Did you passage the cells?
- If available, send photographs of your culture to JCRB by email.