Bio-Rad Apoptosis Assays - Manual (Page 20)

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c. Cultured Cells
i. Collect the cells (5 x 10 6 to 2 x 10 7 ) by centrifugation at 4°C for
5 min at 500 x g. Discard supernatant.
ii. Wash the cells in 1–2 ml of ice-cold PBS. Centrifuge at 4°C for 5
min at 500 x g. Discard supernatant.
iii. Add 750 µl LDB to the pellet, resuspend the cells by pipetting up
and down 10–15x, and vortex for 5 sec.
iv. Incubate the suspension on ice for 30 min.
v. Homogenize using with a Dounce homogenizer.
vi. Transfer the homogenate to a 1.5 ml microcentrifuge tube.
4. Centrifuge the homogenized tissue at 4°C for 10 min at 10,000 x g.
5. Carefully remove the supernatant with a pipet and transfer to a
prechilled 1.5 ml microcentrifuge tube. Label as total extract.
6. Aliquot samples at appropriate volumes into labeled tubes and store at
–80° C. Prepare an aliquot for protein determination.
7. For sample analysis, bring samples to final concentrations of 250 µg/ml
for Bio-Plex Pro RBM Apoptosis Panel 1 and 500 µg/ml for Apoptosis
Panels 2 and 3 by diluting with LDB. Additional dilutions may be
required depending on sample.
6. Run the Assay
Considerations
n
Bring all assay components and samples to room temperature before use
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Use calibrated pipets and pipet carefully, avoiding bubbles. Use new
pipet tips for every volume transfer
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Pay close attention to vortexing, shaking, and incubation instructions.
Deviation from protocol may result in low assay signal and assay variability
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Assay incubations are carried out in the dark on a shaker at 850 ± 50 rpm.
Cover the plate with a plate seal and protect from light with aluminum foil