Frequently Asked Questions - PBMCs
To avoid the risks of contamination and to preserve the integrity of the sample, cryovials should be stored in the vapour phase above liquid nitrogen.
To prevent cell-cell interactions, the initial wash solution may be modified to consist of HBSS (without calcium and magnesium), 10% FBS and 2mM EDTA. If there appear to be stringy fragments in the solution, it may be due to dying cells which release DNA into the media. DNase I, added at final concentration of 0.1 mg/mL (or 200 Kunitz units/mL) and incubated at room temperature for 15 minutes, will reduce the tendency for cells to stick together.
Some cells prefer to be in close contact with each other in culture. The appropriate plate or flask size will vary depending on the number of cells frozen in the cryovial. It is recommended that thawed cells be plated at a high density to optimize recovery. If necessary, try transferring the culture to a smaller flask until the cell density increases.
The freezing and thawing process is stressful to most cells. Be sure to handle the cells very gently. Do not vortex, bang the vial to dislodge the cells, or centrifuge the cells at high speeds. In addition, decrease the time required to thaw the cells as prolonged exposure to cryopreservative can be toxic to the cells.