The role of scavenger receptor BI in hepatitis - eTheses Repository ...

2.4 Basic techniques.
Flow cytometry.
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1. Cells of interest were trypsinised (Gibco) for 5 mins, resuspended in
appropriate media and counted using a haemocytometer. Cells were
then pelleted in a 5804R centrifuge (Eppendorf, Germany) at 1500rpm
for 5 mins and diluted to 2x10 6 cells/ml in PBS + 0.5% bovine serum
albumin (BSA) (Sigma-Aldrich).
2. If cells required fixation prior to staining they were treated for 5 mins
with 3% paraformaldehyde (PFA) (TAAB, UK), followed by a PBS wash
and resuspension in PBS + 0.5% BSA. If permeablisation was
necessary, fixed cell were resuspended in PBS + 0.5% BSA + 0.5%
saponin (Sigma-Aldrich) and all subsequent steps carried out in this
buffer.
3. Suspended cells were incubated for 20 mins at RT to block. If using
saponin this also served as a permablisation step.
4. Antibody staining was performed in a 96 well U bottomed plate
(Corning, NY, USA) with 2x10 5 cells/well. 100$l of cell suspension was
put into each well, the cells were pelleted by centrifugation at 1500rpm
for 5 mins and then resuspended in 70-100ul of primary antibody or
control diluted in PBS + 0.5% BSA (+ 0.01% saponin).
5. After 45 mins incubation at RT, cells were washed with PBS; 100$l
PBS was added to each well and the cells were pelleted by
centrifugation. This process was repeated after which the cells were