Morphology of B cell spots measured directly ex vivo and after polyclonal stimulation. The histogram shows the differences in the distribution of B cell spots measured directly ex vivo (black bars) or after 96 h of polyclonal stimulation (colored bars) in ELISPOT assays. The dashed lines indicate the spot size means in the two groups. A total of 524 spots were analyzed directly ex vivo compared to 2307 spots after polyclonal stimulation using the ImmunoSpot® software version 5.1.36 Professional DC. The images show representative wells for assays performed directly ex vivo or after polyclonal stimulation. The numbers in the left upper corner indicate the spot counts for the two individual wells. The mean spot size measured directly ex vivo was 0.008 mm2 compared to 0.033 mm2 after polyclonal stimulation. Minimum spot sizes were comparable. The maximum spot size was 0.070483 mm2 in direct ex vivo assays compared to a size of 0.442321 mm2 measured after polyclonal stimulation.