Bio-Rad Bio-Plex Pro™ Magnetic Cell Signaling Assays - Manual (Page 11)

9
Adherent Cells
1. Stop the treatment reaction by aspirating the culture medium and
quickly rinsing the cells with ice-cold cell signaling cell wash buffer
(bottle with the blue cap). The volume of buffer required is the same
as the volume of aspirated cell culture medium. Keep the cells on ice
during all steps when possible.
2. Completely remove the buffer before lysing the cells.
3. Immediately add the cell lysis buffer to the cells. The amount of
lysing solution needed depends on the cell density in the culture
vessel (for example, add 1.5–2 ml of lysis buffer to a 10 cm dish that
is ~80% confluent).
Note: It may be necessary to lyse the samples with different volumes of
cell lysis solution to obtain the specified protein concentration range.
4. Scrape the cells with a cell scraper, collect cell suspension into an
appropriately sized tube and gently rock for 20 min at 4°C.
5. Perform either of the following to remove insoluble cellular particulates:
n
Centrifuge the cell lysate solution at 4,500 x g for 20 min at 4°C,
and then filter the lysate using a 0.45 μm syringe filter
n
If the lysate volume is not adequate for filtration, centrifuge the lysate
at 15,000 x g for 10 min at 4°C using a benchtop microcentrifuge
6. Collect the filtered lysate or supernatant after centrifugation.
7. Measure protein concentration using Bio-Rad’s DC ™ protein assay
kit and if needed, adjust protein concentration with cell lysis buffer
containing PMSF and cell lysis factor QG.
8. The suggested working protein concentration range for Bio-Plex ®
cell signaling assays is 3–200 μg/ml (0.15–10 μg per assay well).
9. Store the aliquoted lysates at –70°C until ready to use.