Molecular Devices FLIPR Tetra, User Manual

Page: 279 / 302

FLIPR
®
Tetra High Throughput Cellular Screening System User Guide
0112-0109 H 279
Subtract Bias
The subtract bias algorithm determines the change in fluorescence over
background fluorescence. The user determines the sample number
chosen as background. (This algorithm, by default, is always switched
on and subtracts from Sample 1.) The choice in the sample number to
use as background depends on the assay conditions. For example, the
addition of agonist may dilute the fluorescence signal in the
supernatant, which would appear as a drop in fluorescence. In this
case, the best choice for background is the sample taken at the bottom
of this drop in fluorescence.
Determining Subtract Bias
In our example, the percent positive fluorescence at Sample 1 is
around 20% for all wells. Subtracting the background at Sample 1
would make the data easier to interpret.
Sample 1 (or any sample chosen by the operator) from each well is
subtracted from the samples from the same well (see below)
4 20 23 21 20 115 113 100 119 114 130
5 25 22 21 20 92 93 82 108 108 118
6 30 23 20 20 75 78 68 102 110 118
Sample Time
Well
A1
– Ctrl
A2
– Ctrl
A3
– Ctrl
A4
Exp
A5
Exp
A6
Exp
A7
+ Ctrl
A8
+ Ctrl
A9
+ Ctrl
Note: All wells, at samples 1–3, range from 20 to 23%. The positive
control wells at Sample 4 range from 114 to 130% while the negative
control wells remain at around 20% throughout the experiment.
Time Well
A1
– Ctrl
A2
–Ctrl
A3
– Ctrl
A4
Exp
A5
Exp
A6
Exp
A7
+ Ctrl
A8
+ Ctrl
A9
+ Ctrl
1 0
2 5 1
3 10 1 1 1
4 20 1 94 92 79 98 93 109
5 25 1 71 72 61 87 87 97
6 30 1 54 57 47 81 89 97