9. Add fluorochrome-conjugated antibodies at the optimal concentrations to each sample and incubate for 30 min at 4
C in
the dark (see Note 15).
10. Wash cells by adding 1 mL staining buffer and centrifuging at
500 Â g for 5 min at 4
C.
11. Remove supernatant and resuspend in 500 μL staining buffer
prior to sample acquisition on the flow cytometer (see Note
16). Alternatively, cells can be fixed using Cytofix™ fixation
buffer after removing supernatant if acquisition cannot be
performed within 24 h (see Note 17).
12. Identify immune cell types on the flow cytometer according to
cell surface marker expression (Fig. 2a–d) (see Note 18).
4 Notes
1. SLA is routinely used as a model adjuvant in our immunization
studies. For testing other adjuvants, dose response studies must
be performed to determine the optimal magnitude/quality of
local or systemic immune responses.
2. OVA is routinely used as a model antigen in our immunization
studies. The Alexa Fluor 647-NHS Ester dye is conjugated to
OVA according to manufacturer guidelines [3]. The dye powder is first solubilized in DMSO to a final concentration of
10 mg/mL. A fivefold molar excess of the dye in 10% (v/v)
carbonate buffer (pH 9.3) is then added to OVA in PBS
(pH 7.4) while mixing. The reaction is incubated for 2 h at
room temperature with slow mixing prior to removal of
unreacted dye with an Amicon
® Ultra-4 Centrifugal Filter
Unit with a 10k cutoff membrane. Lastly, the dye-labeled
OVA is resuspended in PBS (pH 7.4). This labeling procedure
yields an optimal dye/protein ratio of approximately 1–2. For
testing other antigens, dose response studies must be performed to determine the optimal magnitude/quality of local
or systemic immune responses.
3. The appropriate negative control for vaccine formulations is
the diluent used for the antigen. PBS is routinely used as the
diluent for OVA in our immunization studies and therefore
would be included as negative control. Similarly, adjuvant
without antigen can be included as an additional negative
control.
4. The specific fluorochromes and viability dye color selected for
the staining panel should be customized to be compatible with
the configuration of the flow cytometer to be used.
Cell Recruitment and Antigen Uptake and Distribution
519
C in
the dark (see Note 15).
10. Wash cells by adding 1 mL staining buffer and centrifuging at
500 Â g for 5 min at 4
C.
11. Remove supernatant and resuspend in 500 μL staining buffer
prior to sample acquisition on the flow cytometer (see Note
16). Alternatively, cells can be fixed using Cytofix™ fixation
buffer after removing supernatant if acquisition cannot be
performed within 24 h (see Note 17).
12. Identify immune cell types on the flow cytometer according to
cell surface marker expression (Fig. 2a–d) (see Note 18).
4 Notes
1. SLA is routinely used as a model adjuvant in our immunization
studies. For testing other adjuvants, dose response studies must
be performed to determine the optimal magnitude/quality of
local or systemic immune responses.
2. OVA is routinely used as a model antigen in our immunization
studies. The Alexa Fluor 647-NHS Ester dye is conjugated to
OVA according to manufacturer guidelines [3]. The dye powder is first solubilized in DMSO to a final concentration of
10 mg/mL. A fivefold molar excess of the dye in 10% (v/v)
carbonate buffer (pH 9.3) is then added to OVA in PBS
(pH 7.4) while mixing. The reaction is incubated for 2 h at
room temperature with slow mixing prior to removal of
unreacted dye with an Amicon
® Ultra-4 Centrifugal Filter
Unit with a 10k cutoff membrane. Lastly, the dye-labeled
OVA is resuspended in PBS (pH 7.4). This labeling procedure
yields an optimal dye/protein ratio of approximately 1–2. For
testing other antigens, dose response studies must be performed to determine the optimal magnitude/quality of local
or systemic immune responses.
3. The appropriate negative control for vaccine formulations is
the diluent used for the antigen. PBS is routinely used as the
diluent for OVA in our immunization studies and therefore
would be included as negative control. Similarly, adjuvant
without antigen can be included as an additional negative
control.
4. The specific fluorochromes and viability dye color selected for
the staining panel should be customized to be compatible with
the configuration of the flow cytometer to be used.
Cell Recruitment and Antigen Uptake and Distribution
519