Running an Experiment
190
0112-0109 H
plates), or 3
μ
L (1536-well plate) per well. Final concentration in
the well should be 5–10
μ
M.
4.
Incubate the cells for 4–6 hrs at room temperature (22 °C or
lower). Cover the plates with aluminum foil to protect from light
and to avoid degradation of coelenterazine.
Coelenterazine Loading for Suspension Cell Assays
1.
Before the day of the assay, remove the selection Antibiotics
from cells.
2.
On the day of the assay, wash cells in seeding flask with 20 mL
DPBS (without Calcium and Magnesium).
3.
Dislodge the cells by adding 15 mL Versene, incubating for 5
minutes in 37 °C
5% CO
2
and immediately adding 20 mL of growth media without
selection antibiotics.
4.
Centrifuge in 50 mL tube @ 1000 RPM or 168g for 2.5–3
minutes.
5.
With supernatant removed and cells re-suspended in 40 mL
BSA-medium, cells should be counted and density adjusted to
5.0 x 10
6
cells/mL.
6.
Under reduced lighting conditions, recommended type of
coelenterazine should be added to reach a final concentration of
5
μ
L.
7.
Cells should be placed in 15 mL aluminum wrapped tubes on
rotating wheel
(~7–8 RPM) at room temp (below 22 °C) for 4 hours to
overnight.
8.
After incubation, cell density should be adjusted while protecting
from light and cells should be placed in the cell stir flask and
stirred on FLIPR
®
Tetra System at speed 6.
9.
Cells should spin protected from light for 1–2 hours prior to the
start of the assay.
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...
