FAQ keywords:
(See FAQs below)

Antibiotics FAQ 3
Cell count FAQ 6
Clonal assay FAQ 13
Dissociation enzyme FAQ 18
Markers FAQ 12
Media color FAQ 7
MEF FAQ 5, 11
Protocols FAQ 1, 2, 9, 10
Quality control FAQ 12, 14
Serum FAQ 16, 17
Splitting FAQ 22
Thaw viability FAQ 4, 8
Transfection FAQ 19

1. Where can I find written protocols? We now enclose a Basic NSCB hES Cell Culture Protocol booklet in all shipments. This booklet includes protocols for thawing, passaging, maintaining, and freezing hES cells. Additional protocols are available here.  Included are the Standard Operating Procedures (SOPs) for the NSCB, the WiCell course protocols, and protocols from ES Cell International, Novocell, University of California- San Francisco, and Technion. There is also a  Practice Live Culture Protocol included with live hES cell shipments. 2. I have previously used human embryonic stem cells obtained from another source or in collaboration with another researcher. Do I still need to read your protocols, or can I jump right in? You should read the provided protocols thoroughly. Make sure you understand them before you thaw your first vial of human embryonic stem cells, even if you had experience with other types of embryonic stem cells from another source or collaborative effort. You may not recover viable cultures post-thaw using other culture protocols. Any changes to the protocols supplied by NSCB are made at the user's own risk. If you have questions about the protocols, please contact the NSCB's technical staff by logging into the website and accessing the request technical support page. You will usually receive a response within 24 hours. For additional support, WiCell provides hands-on training at our facility. 3. Can I add antibiotics to my HES cell media? We do not recommend routinely adding antibiotics to your cell culture media. Adding antibiotics may mask contamination, creating more problems than it solves. 4. When I thaw my cells, there are not as many live cells as I expected based on my experience with other cell types. Does this mean I received a bad vial? Each vial contains an estimated 1x106 cells. Viability from thaw is low and commonly ranges from about 15-100 colonies per vial. The cells that survive thaw may not be initially apparent but will be visible as colonies within about a week and can be passaged by either non-enzymatic or enzymatic passaging usually 7-14 days after thaw. If there are greater than 50 colonies, use enzymatic passaging, if there are less, use non-enzymatic (pick-to-keep) passaging. Both are outlined in the protocol booklet. 5. Can I use STO, 3T3, or some other immortal mouse embryonic fibroblast cell line as feeder cells for my HES cells? Can I use some other mouse strain to produce primary MEF? Can I use primary MEF that I ordered from a commercial vendor? We strongly recommend that you use primary mouse embryonic fibroblasts derived from CF-1 mice exactly as described in the NSCB protocol (SOP-CC-006).  Other sources of fibroblasts could also work, but they should be tested to ensure they support hES cells before use. Any change in fibroblast should be attempted at your own risk. 6. Can I count my HES cells with a hemacytometer? You can count your human ES cells with a hemacytometer, but be advised they are similar in size to disassociated MEF feeder cells. 7. My medium looks too acidic, based on the phenol-red shade of yellowish-orange it turns when completed with serum replacer and non-essential amino acids. Should I be worried? Complete hES Cell Culture Medium is normally a light orange, or "straw-colored" shade. The pH often drops below 7.0 after some time in the incubator and looks nearly yellow. While the pH may seem low, this medium has tested best for undifferentiated HES cell proliferation after extensive screening. We continue to work on media optimization to improve results, but currently the best results are obtained in this medium as formulated.