- Paraformaldehyde is a carcinogen, therefore gloves and a mask
should be worn when preparing this solution. - Removing serum in this way places cells into a quiescent state,
thereby reducing background signalling. In the case of
HUVECs and 3T3-L1 adipocytes, cells should not be quiesced
for any longer than 7 h, as significant apoptosis occurs after
this time. - Use serum-free DMEM when quiescing fibroblasts and
adipocytes. - KRH is a simple buffer that reduces background signalling
even further and abrogates the need for an incubation in a
CO 2 atmosphere. It is important to let cells adjust to this
transition to another buffer prior to adding test substances.
This step can be omitted and experiments continued untilstep
9 if required, although all incubations insteps 1– 8 would need
to occur in a CO 2 incubator. - Add the smallest volume of a concentrated stock that is feasible,
thereby minimizing the volume of DMSO being added. - It is not essential that the test substances such as IL-1β, DMSO,
and A769662 are sterile; however, any steps involving washing
with Medium 199 or DMEM should be performed in a cell
culture hood so as not to contaminate the medium for future
use. Using a repeat pipette when adding compounds/cytokines
will also improve speed and accuracy. - In the case of 3T3-L1 adipocytes, fix for 30 min.
- Take a 21 G needle, and bend the very tip using a pair of
forceps. This, in combination with fine hooked forceps (i.e.,
one in each hand), allows you to easily maneuver the coverslip
in and out of the plate. - Fixed cells can be stored at 4C in 0.5 ml/well PBS at this point,
or the protocol can be continued as described fromstep 13. - For 3T3-L1 adipocytes, simultaneously permeabilize and block
cells in 400μl/well of PBS supplemented with 2% (w/v) BSA,
0.1% (w/v) Saponin and 20 mM glycine for 20 min. Adipo-
cytes have cholesterol-rich membranes; therefore saponin is an
effective detergent to permeabilize membranes as it selectively
removes cholesterol. After this, go tostep 16and follow the
protocol from there. - The serum used to block nonspecific interactions should be
from the animal the secondary antibody is raised in. If using
multiple antibodies for co-staining, ensure that no uninten-
tional cross-reactions can occur between primary and second-
ary antibodies.
316 Sarah J. Mancini and Ian P. Salt