Cell Culture Freezing Media

Determining Viability for a Range of Cell Lines

All cells were frozen and thawed as per the Specialty Media Cell Culture Freezing Media Protocol. Cells were stored in liquid nitrogen, thawed quickly in a 37°C circulating water bath, diluted into complete media, pelleted for 5 minutes at 1000 rpm at 4°C, resuspended in complete media, and plated into the appropriate size culture vessel.

Viability is determined as follows: 16 hours post thawing and plating suspension cell cultures are pelleted, diluted into a trypan blue solution, counted on a hemocytometer and scored for trypan positive and negative cells. These totals are compared to the total number of cells frozen down, and a % viability calculated. Adherent cells are allowed to plate for 16 hours at which time the media is collected and centrifuged to collect the “floating cells” that do not plate. The adherent cells are dissociated, resuspended in complete media and gently pelleted. The “floating cell” pellet and the adherent cell pellet are resuspended in a trypan blue solution and combined. The cells are counted on a hemocytometer and the % viability is determined as described for suspension cells.