Running an Experiment
166
0112-0109 H
3.
Perform a
Protocol Signal Test
to evaluate the basal
fluorescence signal. The desirable fluorescence signal for
intracellular calcium assays is 200–1,200 fluorescence counts
for EMCCD camera and 500–5000 for ICCD camera.
When running a membrane potential assay, the basal
fluorescence counts should be between 1,500–2,500 counts for
standard, EMCCD camera or 10,000–15,000 for aequorin ICCD
camera to allow for a decrease or increase in the fluorescence
signal.
4.
If the basal fluorescence signal is substantially out of these
ranges, the excitation intensity, exposure time and gain or gate
can be adjusted. Use the suggestions below to adjust the basal
fluorescence.
Basal fluorescence too low:
•
Increase the
Excitation Intensity
if the basal fluorescence
signal is too low. During the assay, the
Excitation Intensity
should range between 20% and 100%.
•
Increase the Exposure Time. If you increase the
Exposure
Time
, the read interval will have to be increased to a minimum
of exposure time + 0.1 s (it takes 0.1 s to integrate the data).
•
Increase the
Camera Gain
(EMCCD camera). The gain can vary
between 1 (extremely bright fluorescence) to 240
(luminescence).
•
Increase the
Gate Open
(ICCD camera). The gate can vary
between 0.06% (extremely bright fluorescence) and 35% (very
dim fluorescence).
Camera Type
Calcium Assay
Membrane Potential
Assay
EMCCD
ICCD
EMCCD
ICCD
Excitation wavelength
(nm)
470–495
470–495
510–545
510–545
Emission Wavelength
(nm)
515–575
515–575
565–625
565–625
Excitation Intensity
(%)
50
50
50
50
Exposure Time (s)
0.4
0.533
0.4
0.533
Camera Gain
130
2000
50
2000
Gate Open
—
6%
—
6%
Summary of Contents for FLIPR Tetra
Page 1: ...FLIPR Tetra High Throughput Cellular Screening System User Guide 0112 0109 H December 2011...
Page 12: ...Contents 12 0112 0109 H...
Page 16: ...System Overview 16 0112 0109 H...
Page 40: ...System Hardware Features 40 0112 0109 H...
Page 148: ...Exchanging Hardware 148 0112 0109 H...
Page 156: ...Calibration and Signal Test 156 0112 0109 H...
Page 196: ...Running an Experiment 196 0112 0109 H...
Page 232: ...Robotic Integration 232 0112 0109 H The following drawings illustrate these requirements...
Page 282: ...Data Processing Algorithms 282 0112 0109 H...
Page 294: ...Consumables and Accessories 294 0112 0109 H...
Page 298: ...Using AquaMax Sterilant 298 0112 0109 H...
Page 302: ...Electromagnetic Compatibility EMC 302 0112 0109 H...