In 4 experiments testing 52 plates in an IFNγ assay, well locations were analyzed to determine if they affected spot counts. Outer wells were compared to inner wells, and outer wells had slightly higher average spot counts, around 1.3% higher. When comparing outer, marginal, and core wells specifically, no significant differences in spot counts were found between these groups. No blank wells were observed across the 52 plates and 4,992 total wells tested.

1. Purpose:
In these experiments, we set out to see if well locations had any effects on spot counts and to see if there was a reason why some
people experience empty wells. We compared outer wells to all other wells and then we took a closer look to see if there was a
difference between outer wells, core wells and marginal wells.
Methods:
In 4 separate experiments, 52 plates were tested in a standard IFNγ assay for 24 hours. Each plate had between 12,500 and 100,000
cells plated in 200ul of test media with CMV, pp65 to elicit a response. All spots were counted using an ImmunoSpot reader and
applying the same counting parameters to each well.
Plate layout: Percent difference
Developer A Developer B Developer A Developer B
% difference in count averages
100,000 100,000 100,000 100,000 100,000 100,000 25,000 25,000 25,000 25,000 25,000 25,000 40.00
30.00
100,000 100,000 100,000 100,000 100,000 100,000 25,000 25,000 25,000 25,000 25,000 25,000
20.00
100,000 100,000 100,000 100,000 100,000 100,000 25,000 25,000 25,000 25,000 25,000 25,000 10.00
0.00
100,000 100,000 100,000 100,000 100,000 100,000 25,000 25,000 25,000 25,000 25,000 25,000 0 50 100 150 200 250 300 350 400
-10.00
12,500 12,500 12,500 12,500 12,500 12,500 50,000 50,000 50,000 50,000 50,000 50,000 -20.00
-30.00
12,500 12,500 12,500 12,500 12,500 12,500 50,000 50,000 50,000 50,000 50,000 50,000 -40.00
12,500 12,500 12,500 12,500 12,500 12,500 50,000 50,000 50,000 50,000 50,000 50,000
Out of 52 plates and 360 comparisons, 56% of the time an
12,500 12,500 12,500 12,500 12,500 12,500 50,000 50,000 50,000 50,000 50,000 50,000
outside well had an average that was higher than an inside
well. However, the average spot count only increased in
Purple- outer wells
those outside wells by an average of 1.33%
Green- inner wells
Core wells vs. marginal and
Plate layout
outer wells
600
Developer B Developer A
500
1 2 3 4 5 6 7 8 9 10 11 12
400
A 100,000 100,000 100,000 25,000 25,000 25,000
300 core
B 100,000 100,000 100,000 25,000 25,000 25,000
marginal
C 100,000 100,000 100,000 25,000 25,000 25,000 200 outer
D 100,000 100,000 100,000 25,000 25,000 25,000 100
E 12,500 12,500 12,500 50,000 50,000 50,000 0
100000 cells B 25000 cells A 12500 cells B 50000 cells A
F 12,500 12,500 12,500 50,000 50,000 50,000
developer developer developer developer
G 12,500 12,500 12,500 50,000 50,000 50,000
H 12,500 12,500 12,500 50,000 50,000 50,000
There is no significant spot count
Green- outer wells difference between core wells, marginal
Red- marginal wells
Blue- core wells wells and outer wells.
52 Plates- no blank wells seen: Conclusions:
• In 52 plates and 4,992 wells, not a single
blank well has been seen.
• It is seen that outer wells have higher spot
counts than the inner wells, however the
spot count difference was only 1% higher.
• When comparing outer wells to marginal
and core wells, no spot count difference is
seen between the three groups.