Cytofix cytoperm reagent

Cells were labelled with allophycocyanin (APC) CD11c (HL3), BV421 CD11c (HL3),
FITC CD80 (16-10A1), CD86 PE (GL1), APC CD40 (1C10), PerCP-eFluor 710 CD40
(1C10), PE-Cy7 CD19 (1D3), PerCP-Cy5.5 (H1.2F3), APC TCRβ (H57-597),
phycoerythrin (PE) TCRβ (H57-597), FITC CD3 (145-2c11), APC-eFluor 780
major histocompatibility complex II (M5/114.15.2), BV605 CD45.1 (A20), BV786
CD45.2 (104) and APC IFNγ (XMG1.2) purchased from eBioscience or BD
Pharmingen. pS6 analysis used phospho-S6 Ser 235/236 (Cell Signaling
Technologies) and secondary was PE-conjugated donkey anti-rabbit immunoglobulin
G (Jackson ImmunoResearch). 2-NBDG (Life Technologies) was added to cells at
35 μM for 1 h prior to analysis. Live cells were
gated by forward scatter (FSC-A) and side scatter (SSC-A) analysis. Single cells
were selected by FSC-A and FSC-W analysis. For intracellular staining, cells
were then fixed and permeabilized using Cytofix/Cytoperm reagent (BD
Pharmingen). For cytokine analysis, endocytosis was blocked using golgi plug (BD
Pharmingen) for 4 h. Gating strategies for all flow cytometry
analysis are outlined in Supplementary
Figs 5 and 6. Data were acquired on a FACSCanto or LSRFortessa (Becton
Dickinson) and analysed using the FlowJo software (TreeStar).

Cells were fixed/permeabilized using the Cytofix/Cytoperm reagent (100 µl per million cells, BD Pharmingen) at RT for 20 min, and washed twice with 1x Permwash buffer (BD Pharmingen) prior to immunofluorescent antibody labeling. Washed cells were pelleted by centrifugation at 400 × g, 4 °C for 5 min and resuspended in 1x Permwash buffer containing fluorophore-conjugated DR-, DM- DO-, and CLIP-specific antibodies (~1 µg of each antibody per million cells per 100 µl buffer) and incubated on ice for 1 h. Antibody-labeled cells were washed twice with 1x Permwash buffer and then resuspended in PBS + 1% BSA for analysis on a flow cytometer, either FACSCalibur or LSRII (BD Biosciences). Flow cytometric data were analyzed using FlowJo software (Tree Star, Inc.).

Cells were fixed and permeabilized with the BD Cytofix/Cytoperm reagent for 20 min at room temperature. Cells were washed with BD Perm/Wash buffer and then incubated with mouse anti-human SPG7 monoclonal antibody (dilution 1/10,000; OriGene) or the IgG1 isotype control for 1 h. Cells were washed and incubated with Alexa Fluor secondary antibody for 30 min. Cells were washed, and the relative fluorescence intensity was measured for 10,000 events per sample on a BD LSRFortessa, and the data were analyzed using BD FACS Diva software. Mean fluorescence intensity was calculated as the fluorescence intensity of the paraplegin antibody to the intensity of the isotype control antibody.

Inflammasome complex assembly was evaluated by the detection of “specks" formation using immunofluorescence microscopy. MDM were fixed and permeabilized with Cytofix/Cytoperm reagent (BD Biosciences) for 30 min at 37°C and 5% CO₂ and incubated with primary antibody for NLRP3 (1:100 mouse anti-human NLRP3, Abram) and/or NLRC4 (1:200 rabbit anti-human NLRC4; Biolegend) overnight at room temperature. Fluorescent secondary antibodies (Alexa 488-conjugated goat-anti-mouse IgG1, or Alexa 647-conjugated goat-anti-rabbit IgG1; Thermofisher Scientific) were then added for 1 h. 4',6-Diamidine-2'-phenylindole dihydrochloride (DAPI; Sigma-Aldrich) was used for nuclear counterstaining. Image acquisition was performed at the microscope facility at the Laboratory of Cellular Biology from the Butantan Institute (São Paulo, SP, Brazil) using a DMi8 confocal laser scanning microscope equipped with a digital camera DFC310 FX (Leica). The counting of NLRP3+ and NLRC4+ specks in MDM was performed manually by observing speck formation within the cells in 10 fields (26 (link)).

For intracellular cytokine staining of YF-tetramer⁺ CD8⁺T-cells, PBMCs of tetramer reactive samples were stimulated for 6 hours with phorbol myristate acetate (PMA) and ionomycin. One to two million cells were incubated in medium consisting of RPMI-1640 with 10% fetal calf serum (FCS) in the presence of PMA (10 ng/mL) and ionomycin (1 ng/mL), anti-CD107a FITC (eBioscience), brefeldin A (10 microg/mL; Invitrogen), GolgiStop (BD Biosciences) and co-stimulation (anti-CD28) for 6 hours at 37°C and 5% CO2. As a control, the same conditions without PMA and ionomycin were used. After incubation, 20 μL of tetramer mix was added to the samples in a 96-wells plate for 30 minutes at 4°C. Subsequently, 30 μL of antibody mix with CD3 V500 (Invitrogen) and CD8 BV785 from Biolegend were added for 30 minutes. The Cytofix/Cytoperm reagent (BD Biosciences) was used for fixation and permeabilization. After permeabilization, the following monoclonal antibodies were added: anti-TNF-α AF700, anti-IL-2 PE (BD Biosciences), anti-Mip1-β PE-Cy7 (Biolegend), anti-IFN-γ APC-eFluor 780 (eBiosciences). Cells were analyzed by LSR Fortessa and FlowJo v. 9.7.6 software (Stanford University, 1995–1996).

PBMCs were collected 1 week after the last drug/peptide injection. Erythrocytes were lysed in ammonium chloride-potassium bicarbonate buffer, and leukocytes were pulsed ex vivo with relevant peptide (1 μg/ml) (e.g., E7 aa49–57, Ova aa258–265, or AH1 aa6–14) overnight in the presence of Brefeldin A (BD Biosciences). Cells were stained with PE-labeled α-CD8 mAb (BD Biosciences), fixed and permeabilized with Cytofix/Cytoperm reagent (BD Biosciences), and then stained with FITC-labeled anti-IFN-γ mAb (BD Biosciences). The frequency of IFN-γ⁺ CLTs was examined by flow cytometry via FACSCalibur device (BD Biosciences), as previously described [28 (link)]. For tetramer binding analysis, PBMCs were co-stained with FITC-labeled anti-CD8 mAb (BD Biosciences) and PE-labeled H-2D^b tetramer loaded with HPV-16 E7 epitope (aa49–57; RAHYNIVTF) (Beckman Coulter, Hialeah, FL), and then examined by flow cytometry. For analysis of tumor-infiltrating E7-specific CTLs, tumor tissue was excised from tumor-bearing mice, minced, and passed through a 100 μm strainer. Single cells were co-stained with FITC-labeled α-CD8 mAb and PE-labeled E7-D^b tetramer and examined by flow cytometry. All data analysis was performed on gated lymphocyte populations (as defined by FSC/SSC features) using FlowJo software (Tree Star, Ashland, OR).