Violet live dead stain kit

For the analysis of the magnitude and phenotype of the HIV-1 gp140-specific Tfh cell responses, 4 × 10⁶ splenocytes (erythrocyte-depleted) were seeded on 96-well plates and stimulated for 6 h in complete RPMI 1640 medium (2 mM L-glutamine, 100 units/ml of penicillin/100 μg/ml of streptomycin, 10 mM Hepes, and 0.01 mM β-mercaptoethanol) with 10% FCS, 1 μl/ml Golgiplug (BD Biosciences), anti-CD154 (CD40L)-PE (BD Biosciences), and 5 μg/ml of HIV-1 96ZM651 clade C Env-1+Env-2+Env-3 peptide pools + 5 μg/ml of HIV-1 96ZM651 gp140 protein. Non-stimulated samples (RPMI) were used as control. After stimulation, cells were washed, stained for surface markers, permeabilized (Cytofix/Cytoperm kit; BD Biosciences) and stained intracellularly. Dead cells were excluded using the violet LIVE/DEAD stain kit (Invitrogen). For the analysis of Tfh cells, the following fluorochrome-conjugated antibodies were used: CD154-PE, IL-4-Alexa488 and IFN-γ-PE-Cy7 for functional analyses and CD4-Alexa700, CD8-V500, PD1(CD279)-APC-efluor780, CXCR5-PE-CF594, and CD44-PE-Cy5 (SPRD) for phenotypic analyses. All antibodies were from BD Biosciences.

To determine the magnitude and phenotype of the HIV-1- or VACV-specific CD4 and CD8 T cell immune responses, 2 × 10⁶ splenocytes (erythrocyte-depleted) were seeded on 96-well plates and stimulated for 6 h in supplemented RPMI 1640 medium with 10% FCS, 1 µL/mL Golgiplug (BD Biosciences, Franklin Lakes, NJ, USA), anti-CD107a-FITC (BD Biosciences) and 5 µg/mL of HIV-1 clade C ZM96 gp140 (Env-1 + Env-2 + Env-3) or Gag peptide pools or 1 µg/mL of HIV-1 clade C CN54 Pol-Nef (Gag-Pol + Pol-1 + Pol-2 + Nef) peptide pools or 10 µg/mL of VACV E3 peptide. After stimulation, the splenocytes were washed, stained for surface markers, permeabilized (Cytofix/Cytoperm kit; BD Biosciences), and stained intracellularly with appropriate fluorochromes. For the analysis of HIV-1- and VACV-specific CD4 and CD8 T cell responses, the following fluorochrome-conjugated antibodies were used: CD3-PECF594, CD4-APCCy7, CD8-V500, CD107a-FITC, IL-2-APC, IFN-γ-PeCy7 and TNF-α-PE for functional analyses and CD127-PerCPCy5.5 and CD62L-Alexa700 for the phenotypic analyses. All of the antibodies were from BD Biosciences. The dead cells were excluded using the violet LIVE/DEAD stain kit (Invitrogen).

To analyze the magnitude and phenotype of the HIV-1-specific Tfh cell immune responses, 2 × 10⁶ splenocytes (erythrocyte-depleted) were seeded on 96-well plates and stimulated for 6 h in supplemented RPMI 1640 medium with 10% FCS, 1 µL/mL Golgiplug (BD Biosciences), anti-CD154 (CD40L)-Biotin (BD Biosciences) and 5 µg/mL of HIV-1 clade C ZM96 gp140 (Env-1 + Env-2 + Env-3) or Gag peptide pools or 1 µg/mL of HIV-1 clade C CN54 Pol-Nef (Gag-Pol + Pol-1 + Pol-2 + Nef) peptide pools. After stimulation, the splenocytes were washed, stained for surface markers, permeabilized (Cytofix/Cytoperm kit; BD Biosciences), and stained intracellularly with appropriate fluorochromes. For the analysis of HIV-1-specific Tfh cell responses, the following fluorochrome-conjugated antibodies were used: CD4-Alexa700, CD8-V500, CD154 (CD40L)-Biotin/Avidin-PE, IL-4-Alexa488, IFN-γ-PeCy7 and IL-21-APC for functional analyses and CXCR5-PECF594, PD1 (CD279)-APCefluor780 and CD44-PeCy5 (SPRD) for phenotypic analyses. All of the antibodies were from BD Biosciences. The dead cells were excluded using the violet LIVE/DEAD stain kit (Invitrogen).

To analyze the magnitude and phenotype of the HIV-1- and VACV-specific CD4 and CD8 T cell responses at the different time points analyzed (days 24, 54 and 123), 2 × 10⁶ splenocytes or lymphocytes from inguinal LNs (erythrocyte-depleted) were seeded on 96-well plates and stimulated ex vivo for 6 h in complete RPMI-1640 medium with 10% FCS, 1 μL/mL Golgiplug (BD Biosciences), anti-CD107a-FITC (BD Biosciences) and 5 μg/mL of the HIV-1 clade B consensus peptide pools or 10 μg/mL of the VACV E3 peptide (E3 peptide was only added for the analysis at days 54 or 123). Non-stimulated samples (RPMI) were used as control. After stimulation, cells were washed, stained for surface markers, permeabilized and stained intracellularly. Dead cells were excluded using the violet LIVE/DEAD stain kit (Invitrogen). For the analysis of CD4 and CD8 T cell immune responses, the following fluorochrome-conjugated antibodies were used: IFN-γ-PE-Cy7, IL-2-APC and TNF-α-PE for functional analyses and CD3-PE-CF594, CD4-APC-Cy7, CD8-V500, CD19-SPRD, Gr-1-SPRD and CD49b-Alexa 700 for phenotypic analyses. All antibodies were from BD Biosciences. Cells were acquired in a GALLIOS flow cytometer (Beckman Coulter), and the analysis of the data was performed using FlowJo software (Version 10.4.2; Tree Star). The number of lymphocyte-gated events ranged between 10⁵ and 5 × 10⁵.

Env-specific CD8 T cell responses were analyzed by ICS assay and flow cytometry using previously described protocols (7 (link), 29 (link)). Briefly, 4 × 10⁶ splenocytes or 10⁶ cells from DLNs were stimulated with 5 μg/ml of clade C HIV-1 Env-1 peptide (a H-2^d-restricted CTL epitope with the sequence PADPNPQEM as previously described (30 (link)) along with 1 μl/ml GolgiPlug (BD Biosciences), anti-CD107a-Alexa 488 (BD Biosciences) and monensin (1X; eBioscience), in RPMI 1640-10% FCS at 37°C for 6 h in a 96-well plate. Next, the cells were washed, stained for the surface markers, fixed, permeabilized (Cytofix/Cytoperm kit; BD Biosciences) and mixed with anti-CD16/CD32 (BD Biosciences) for blocking the Fc receptors. Cells were stained intracellularly with antibodies against intracellular cytokines conjugated with the appropriate fluorochromes. Live cells were selected using the violet LIVE/DEAD stain kit (Invitrogen). Cells were stained with the following conjugated antibodies: CD3-PE-CF594, CD4-APC-Cy7, CD8-V500, IFN-γ-PE-Cy7, IL-2-APC, and TNF-α-PE and then acquired through a GALLIOS flow cytometer (Beckman Coulter). Data analysis was carried out with FlowJo (Version 10.4.2; Tree Star, Ashland, OR) and the response to specific stimuli was obtained after corrections made with values obtained in unstimulated control samples.

For intracellular cytokine staining, lymph node T cells or cells after primary ConA activation and IL-2 expansion were stimulated (1 h, 37°C) in medium with phorbol 12-myristate 13-acetate (PMA; 50 ng/ml) and ionomycin (2 μg/ml). Brefeldin A (GolgiPlug, 20 μl/ml; BD PharMingen) was added and cells incubated (3 h, 37°C). After surface marker staining with appropriate combinations of labeled anti-CD4, -CD8, -Thy1, -CD44, and -CD62L, cells were washed with PBS, fixed and permeabilized with Cytofix/Cytoperm (100 μl, 20 min, 4°C), washed once with Perm/Wash buffer and blocked with 150 μl Perm/Wash buffer + 1% BSA (30 min, room temperature (RT)). Anti-IL-17-PE, -IL-2-PE and -IFNγ-PE antibodies were added (all 1/100, PharMingen); permeabilized cells were incubated (20 min, 4°C) and washed twice in Perm/Wash before FACS analysis.
For the vaccinia infection experiment, splenocytes were stained for surface markers with anti-CD3-PE-CF594, -CD4-APC-Cy7, -CD8-V500 (all from BD), fixed, permeabilized (Cytofix/Cytoperm kit; BD), and stained intracellularly with anti-IFNγ-PE, and -TNFa-PE-Cy7 (from BD). Dead cells were excluded using the violet LIVE/DEAD stain kit (Invitrogen).