TEJIENDO UNA ANTROPOLOGÍA RARÁMUR
C APÍTULO III
All data were analysed using Flowjo™ software (Treestar, Oregon).
2.2.10.1
Measurement of cell death
Cell death was assessed using Annexin V and PI staining. For Annexin V staining, all washes, incubations and measurements were performed in 1x Annexin V binding buffer. PI staining was performed in PBS. BMDCs were stimulated for 24 h, then washed and incubated with 1 μg/ml Annexin V-FITC (200 μl). After 5 min, cells were washed and resuspended in 200 μl of binding buffer. Finally, PI was added directly to FACS tubes (1 μg/ml) immediately before acquiring the samples on the BD FACs Canto flow cytometer.
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2.2.10.2
Measuring DC maturation
BMDCs (1x106 cells/ml) were plated on a 96-well tissue culture plate and stimulated with
medium, LPS, CpG or PLGA particles. After 24 h, plates were centrifuged (400g, 5 min, 4oC)
supernatants discarded and BMDCs were resuspended in 200 μl FACS buffer in FACS tubes
(BD Falcon). The cells were again centrifuged (400g, 5 min, 4oC) and stained with Aqua
LIVE/DEAD (Invitrogen) used at a 1:1000 dilution in PBS in a total volume of 200 μl/sample. After 30 min in the dark, cells were washed again and resuspended in 200 μl of FACS buffer supplemented with purified anti-mouse CD16/CD32 monoclonal antibody (dilution outlined in
section 2.1.7) and incubated at 4 oC for 10 min. Cells were stained with Fluorochrome-labelled
anti-CD11c and antibodies specific for the DC maturation markers CD80, CD86, CD40 and MHC class II (section 2.1.7) for 30 min on ice in the dark. Afterwards, cells were centrifuged
(400g, 5 min, 4 oC) and washed twice with 200 µl FACs buffer before being analysed for
immunofluorescence using a BD LSRFortessa flow cytometer.
2.2.10.3
DC and OTII co-culture and staining
BMDCs (2x104 cells/ml) were plated in a 96-well tissue culture plate and stimulated with
medium, LPS, OVA protein (25 µg/ml) or different sized PLGA particles (0.1 mg/ml) alone or in combination with each other overnight. The following day, spleens and lymph nodes from an OTII mice were taken and processed as outlined in section 2.2.8. Cells were counted and T cells were isolated using a MACS protocol from Pan T cell isolation kit II (Miltenyi Biotec). Briefly, LS MACs columns (Miltenyi Biotec) were placed in the magnetic field of a MACS separator and washed with 3 ml of MACS buffer (Section 2.1.1). Cells were resuspended in 40
µl MACS buffer per 107 total cells, 10 µl Pan T cell Biotin-Antibody cocktail per 107 total cells
were added, mixed and incubated for 5 min at 4oC. 30 µl of buffer was added per 107 total cells
and then 20 µl of Anti-Biotin Microbeads per 107 total cells was added, mixed and incubated
for an additional 10 min at 4oC. Cells were then added to the washed LS columns on the MACS
separator and the flow through containing unlabelled cells was collected, representing the enriched T cells. The column was also washed (1x3 ml) with MACs buffer to collect additional
T cells. The T cells were centrifuged, resuspended (1x106 cells/ml) in T cell media and
incubated at 37 oC for 1 h.
T cells were centrifuged (400g, 5 min, 4oC) and resuspended in 1 ml PBS containing 5% (v/v)
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adding 2 µl of a 5 mM stock to 1 ml of PBS and quickly added to 1 ml of thoroughly resuspended cells. Cells were mixed immediately on a vortex and incubated in the dark for 5 min at room temperature. Cells were then washed 3 times by adding 10 ml PBS containing 5%
(v/v) FCS, centrifuged (400g, 5 min, 4 oC) and supernatant discarded. The BMDCs in 96-well
plates were centrifuged and supernatant removed. Simultaneously, T cells were resuspended in
T cell media, counted and 2x105 T cells/well, in a final volume of 250 µl were added to the 96-
well plates containing the BMDCs. The co-culture was left for 72 h.
After 72 h, cells were stained for surface markers and intracellular cytokines. Carefully, 50 µl of media from the co-culture was removed and 50 µl of Brefeldin A (50 µg/ml) in T cell media was added to wells to give a final concentration of 10 µg/ml. Brefeldin A disrupts the Golgi apparatus in cells, restricting the secretion of intracellular cytokines. The 96-well plates were returned to the incubator for 4 h.
The 96-well plates were centrifuged (400g, 5 min, 4 oC) and supernatants discarded. Cells were
resuspended in 200 µl PBS containing 2 mM EDTA and triplicates pooled into the same FACS
tube. Cells were washed with 1 ml PBS, centrifuged (400g, 5 min, 4 oC) and stained with Aqua
LIVE/DEAD (Invitrogen) used at a 1:1000 dilution in PBS in a total volume of 200 μl/sample. After 30 min in the dark, cells were washed again and resuspended in 200 μl of FACS buffer supplemented with purified anti-mouse CD16/CD32 monoclonal antibody (dilution outlined in
Table 2.) and incubated at 4 oC for 10 min. Cells were stained for T cells with Fluorochrome-
labelled anti-CD3 and CD4 (dilutions outlined in Table 2.) for 30 min on ice in the dark.
1 ml of PBS was added to the cell suspension, centrifuged (400g, 5 min, 4 oC) and supernatant
decanted. Cells were resuspended in 100 µl PBS and 100 μl of 2% PFA was added to fix the cells. The cells were vortexed and incubated at room temperature in the dark for 15 min. 1 ml
of PBS was added and the cell suspension was centrifuged (400g, 5 min, 4 oC) and the
supernatant decanted. The cells were resuspended in 50 μl permeabilisation buffer (Table 2.12)
and incubated for 30 min at 4 oC in the dark. Cells were then stained with 50 µl permeabilisation
buffer containing intracellular fluorochrome-labelled antibodies (0.3 μl/sample) and incubated in the dark at room temperature for 30 min. Cells were then washed twice by adding 1 ml PBS,
centrifuging the cells (400g, 5 min, 4 oC) and discarding the supernatants. The cells were
resuspended in 200 μl FACS buffer for analysis of immunofluorescence. Cells were analysed using a BD LSRFortessa flow cytometer.
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