Bio-Rad Bio-Plex Pro™ Magnetic Cell Signaling Assays - Manual (Page 12)

10
Suspension Cells
1.
Collect cell suspension and pellet the cells by spinning at 1,000 x g for
5 min at 4°C.
2. Aspirate off cell culture medium completely.
3. Wash by resuspending the cells with ice-cold cell wash buffer
(bottle with the blue cap) .
4. Centrifuge the cells at 1,000 x g for 5 min at 4ºC.
5. Completely remove the buffer.
6. Immediately add the proper volume of cell lysis buffer and gently rock
for 20 min at 4°C.
7. Remove insoluble cellular particulates as described in Adherent Cells
step 5 above .
8. Follow Adherent Cells steps 6–9 above.
Tissue Samples
1. Cut the tissue into small pieces (~3 x 3 mm) for ease of handling and
blood removal. If necessary, wash the tissue with ice-cold cell wash
buffer (bottle with the blue cap) to completely remove all blood. Then
transfer the tissue to a 2 ml tissue grinder.
2. Add an adequate volume of cell lysis solution and grind the tissue
sample on ice using approximately 20 strokes.
Note: It may be necessary to lyse the samples with different volumes of
cell lysis solution to obtain the specified protein concentration range.
3. Transfer the ground tissue to a clean microcentrifuge tube and freeze
sample at –70°C. Freezing and thawing samples helps increase cell
lysis effects.
4. Thaw the sample and sonicate on ice (for example, with a Sonifier
450: Duty Cycle = 40, Output = 1, Pulse Sonicating = 18x).
5. Remove insoluble cellular matter as in Adherent Cells step 5 above.
6. Follow Adherent Cells steps 6–9 above.