Anti cd45 v500

To assess the quality of T cell isolation, cells were fluorescently stained with anti-CD45-V500, anti-CD3-FITC, anti-CD4-APC-H7, anti-CD8a-PE, anti-CD19-APC, anti-CD14-PerCP, and anti-CD56-PE-Cy7 antibodies (Beckton Dickinson, Franklin Lakes, USA). For monitoring T cell subsets (day 3 to day 8), cells were fluorescently stained with anti-CD3-FITC, anti-CD4-BV510, anti-CD8-PE-Cy7, anti-CD127-AlexaFluor®647, anti-CD25-PE, anti-CD69-APC-H7, and anti-LAG-3-BV421 antibodies (Beckton Dickinson, Franklin Lakes, USA). Briefly, cells were harvested, washed, resuspended in 1× phosphate buffered saline (PBS) at pH 7.4, and incubated with antibodies for 30 min at 4 °C. After antibody incubation, cells were washed with PBS, incubated with 7-aminoactinomycin (7-AAD) reagent 10 min prior to measurement and analyzed via flow cytometry as described below.
All cells were analyzed by flow cytometry using BD FACSCanto™ II (Beckton Dickinson, Franklin Lakes, USA). Dead cells and cell debris were excluded from the analysis based on 7-AAD fluorescence and scatter signals. Analysis was performed using Kaluza flow cytometry analysis software (Beckman Coulter, Brea, USA).

Blood samples were collected by cardiac puncture into EDTA-coated probes. Then, 50 µL of blood sample was incubated with 25 µL of blocking buffer (20% rat serum and 20% FBS in PBS) for 10 min at room temperature (RT). After incubation, surface antigens were stained with the following antibodies: anti-CD11b BV510 (562950; BD Horizon; 1:400), anti-CD45 V500 (562129; BD Horizon; 1:200), anti-Ly6G BV605 (563005; BD Horizon; 1:200) and Ly6C BV711 (128037; BioLegend (San Diego, CA, USA); 1:400) for 30 min at room temperature (RT) in the dark. Subsequently, 500 µL of RBC Easy Lyse 1× solution (S2364; Dako (Glostrup, Denmark)) was added to the samples to lyse erythrocytes, and the samples were lysed for 15 min at 4 °C. After passing the cells through a 100 μm strainer, the percentages of peripheral blood leukocytes subpopulations were analysed with a BD LSRFortessa flow cytometer.

Splenocytes or isolated bone marrow neutrophils were seeded into 96-well plates (1–2 × 10⁶ per well). Cells were centrifuged for 5 min at 350 RCF; then, they were stained with Fixable Viability Stain 440UV (565388; BD Horizon) according to the manufacture’s recommendation and incubated for 6 min at 37 °C, 5% CO₂. After two washes, the samples were blocked with 5% FBS in PBS for 10 min at RT. Subsequently, surface antigens were labelled using the following antibodies: anti-CD11b BV510 (562950; BD Horizon; 1:400), anti-CD45 V500 (562129; BD Horizon; 1:200), anti-Ly6G BV605 (563005; BD Horizon; 1:200), anti-Ly6C BV711 (128037; BioLegend; 1:400), anti-CD80 Pe-Cy7 (104734; BioLegend; 1:200), anti-CD83 APC (558206; BD Pharmingen; 1:200), anti-I-A/I-E BB700 (746197; BD OptiBuild; 1:200) and anti-CD86 FITC (561962; BD Pharmingen; 1:200) for 30 min at RT. Cells were then fixed using BD Cell fix and analysed using a BD LSRFortessa flow cytometer.

B lymphocytes (CD19⁺), CTL (CD3⁺CD8⁺) and Th lymphocytes (CD3⁺CD4⁺) frequencies were assessed using anti-CD19-APC-Cy7 (cat. 557791, BD Biosciences), anti-CD3-APC (cat. 561810, BD Biosciences), anti-CD8-APCH7 (cat. 560273, BD Biosciences), anti-CD4 PerCP-Cy5.5 (cat. 341654, BD Biosciences) and anti-CD45-V500 (cat. 560777, BD Biosciences) on fresh heparinized blood. Then PBMCs were isolated from the remnant aliquot of fresh heparinized blood by Ficoll-Paque (Amersham Bioscience) to assess Th subpopulations. Treg (CD4⁺/CD25^hi/Foxp3⁺) lymphocytes were evaluated using Foxp3 staining kit (cat. 560047, BD Pharmingen; isotype control: cat. 557702, BD Pharmingen), while Th1 (CD4⁺/ IFN-γ⁺) and Th17 (CD4⁺/IL-17A⁺) using Human Th1/Th2/Th17 Phenotyping Kit (cat. 560751, BD Pharmingen), according to manufacturer’s instructions. The last was performed both on basal PBMC and on anti-CD3 (0.166 ng/µl), IL-2 (0.08 pg/µl) stimulated PBMC for 18hs. Gating strategies are shown in Supplementary Fig. 7.
Data were collected on a BD FACSCantoTM II cytometer (BD Bioscience) and analysed using BD FACSDiva™ Software.

The levels of ex vivo Per2 expression was evaluated using whole blood 24 h after draw. Two hundred microliter of whole blood was stained for 20 min at 4°C with surface antibody anti-CD34 APC and anti-CD45 V500 (Becton-Dickinson BD); following monoclonal staining, BD Pharm Lyse (BD Biosciences) lyse solution was used to lyse red blood cells according to standard procedures and fixed with 4% paraformaldehyde. After red cell lysing, cells were incubated with anti-PER2 antibody (ab179813 Abcam) or anti-Sirt1 Alexa Fluor 488 (ab196368 Abcam) for intracellular staining 20 min at room temperature using a permeabilizing buffer containing 0.5% saponin (PBS1X /BSA 1%/NaN3 0.1%/Saponin 0.5%) and then washed with 0.1% saponin buffer. Alexa Fluor 488-coniugated goat anti rabbit (Invitrogen) diluted in staining buffer containing 0.1% saponin was used as secondary antibody. To evaluate cell viability, we used the 7AAD staining. In some experiments 1 mL of whole blood of HIV infected patients was stimulated with different concentrations of Resveratrol (R5010- Sigma-Aldrich) and YK-3-237 (Tocris) overnight at room temperature (see section results for details). After stimulation, the frequency of HPC expressing Per2 was evaluated by flow cytometry. At least 300,000 total cell events were acquired on FACSCanto II (Becton-Dickinson BD) flow cytometer and analyzed using DIVA software.

Spleen and tumor cells obtained from control or treated tumor-bearing mice were thawed, centrifuged, and incubated with monoclonal antibodies conjugated with fluorophores: anti-CD45 V500, anti-CD11b PerCP-Cy5.5, anti-CD11c BV605, anti-CD4 APC, anti-B220 APC, anti-CD49b APC, anti-Ly6C PE, anti-Ly6G APC-Cy7, anti-MHCII FITC, and anti-CD86 PE-Cy7 (all from BD Biosciences). After incubation, cells were suspended in PBS with DAPI dye (Molecular Probes) and analyzed using LSR Fortessa with Diva Software (Becton Dickinson) according to the procedure described by Rossowska and coworkers (27 (link)).