UH_2011
Protocol: Thawing frozen PBMCs
1| Determine the total number of PBMCs needed for the experiment, taking into account the
number of stimulations to be performed, including a negative and positive control well for each
sample. Select cell vials to be thawed accordingly.
2| Thaw cell sample vials in a 37 C water bath until there is a pea-sized pellet of ice remaining.
3| Transfer the cell suspensions to a correspondingly labeled 15-ml conical tube containing
prewarmed culture medium with DNase.
4| Centrifuge at 1300 rpm for 8 min at room temperature (18–28C), aspirate supernatant and
resuspend cells in 3ml culture medium (typically R10 no DNase).
5| Place cells in labeled 6-well plates (3ml max) T-25 culture vials (10ml max) at density of 34x106 PBMCs per ml and culture overnight in an incubator at 37C and 5% CO2.
DNase working solution Dilute DNase stock (Sigma, cat. no. DN-25) to a concentration of
2,000 ug ml–1 with sterile PBS. Divide into aliquots in desired number of vials and store at 4C.
Culture medium with DNase Prepare a 1:100 solution of DNase working solution in culture
medium.