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Reagents
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Pharmingen
anti-CCR5 antibody 2D7-FITC (cat.no. 36464X)*.
Store at 4°C. Use at 1:5 dilution.
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Pharmingen
anti-CXCR4 antibody 12G5-PE (cat.no. 36195X). Store
at 4°C. Use at 1:100 dilution.
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Pharmingen
FITC-conjugated mouse IgG2a, kappa isotype control (cat.no.
20074A). Store at 4°C. Use at 1:100 dilution.
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Pharmingen
PE-conjugated mouse IgG2a, kappa isotype control (cat.no.
20075A). Store at 4°C. Use at 1:100 dilution.
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Falcon
5ml FACS-tubes (cat.no. 2054).
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PBS/1% BSA:
dissolve 4mg Sigma BSA (cat.no. A-2153) in 400ml
PBS. Filter through 0.22 µm filter. Store at 4°C.
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2x
Storage buffer (2% Formaldehyde, 0.05%
Glutaraldehyde): add 5.71 ml 35% Formaldehyde and
200 µl 25% Glutaraldehyde to 94ml PBS. Store at 4°C.
Protocol
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For
each sample to be analyzed use up to 0.5 x 106
cells (see note).
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Pellet
cells in 5 ml FACS-tube (1100 rpm 5 min).
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Resuspend
in 100 µl of cold PBS/1%BSA with appropriate
concentration of specific antibody or isotype
control.
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Vortex
at low speed for 1-2 seconds.
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Incubate
for 30 minutes in the dark at 4°C.
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Add
5 ml of cold PBS/1% BSA and spin (1100 rpm 5 min).
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Remove
supernatant and resuspend cells in sheath fluid for
immediate analysis or resuspend cells in 0.5 ml of
1.0% Formaldehyde (1x Storage Buffer) and store them
at 2-8°C in the dark. Analyze fixed cells within 24
hours.
Note:
for each cell sample that is being tested for chemokine
receptor-expression, be sure to also test control (i.e.
test 0.5 x 106 cells with chemokine
receptor-specific antibody and 0.5 x 106
cells with the isotype control antibody) for aspecific
sticking.
*Also
available: anti-CCR5 antibody 2D7-PE (cat.no. 36465X)
for use on GFP-expressing cells. Isotype control:
Pharmingen PE-conjugated mouse IgG2a, kappa isotype
control (cat.no. 20075A).
For specifics on appropriate handling and waste procedures please see the
online chemical SOPs or our waste and spill notebook located in
room 352.
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