Cell Counting Procedure. After the final split of the SC assay, the confluent cell layer is suspended in 250 m l of PBS, and the cell number is counted in a Neubauer chamber. If many samples are being processed, we count the cells in eight wells of each plate, average the cell density, and dispense the required number of cells (1,000-25,000) based on the average cell count of the sampled wells. To confirm the number of cells actually plated in each well, one may dispense 100 m l of an appropriate dilution of each suspension (to contain 200-1,000 cells) into a separate ELISPOT plate. The cells are drawn onto the membrane by suction, dried for 1 h at 50ºC, and stained with 0.04% Trypan blue in lysis buffer for a few seconds, washed twice with PBS, and counted with the ELISPOT equipment. This procedure allows the accurate determination of spots per number of plated cells to be calculated.