Molecular Devices FLIPR Tetra, Руководство пользователя

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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.