Anti cd4 pe

Brains from 22- to 26-week-old animals were homogenized using a 5-ml Glass Tissue Grinder (A. Hartenstein). The homogenized solution was mixed with 100 % Percoll solution (GE healthcare) to a final 70 % Percoll solution and layered under a 30 % Percoll solution. After 30 min centrifugation at 500g without brake at RT, the cells were isolated from the 30–70 % layer and washed with PBS. Per staining, 100 000 cells were used. The staining was performed in FACS-PBS (PBS, 2 % FCS, 5 mM EDTA) in the presence of the FcBlocker (Miltenyi Biotec) using the following fluorescently labeled antibodies: anti-CD4-PE, anti-CD8-APC, anti-CD11b-FITC, and anti-CD45-APC (all from Miltenyi Biotec), for 15 min at RT in the dark. Samples were analyzed using a Gallios flow cytometer (Beckman Coulter). Thirty thousand events were recorded. Flow cytometry data were analyzed using the FlowJo 8.5.3 software (FlowJo, LLC). For viability testing, the Fixable Viability Dye eFluor^® 780 (eBioscience) was used according to the manufacturer’s instructions. The gating of the living cell population was performed based on the live staining; dead cell populations were excluded. The total cell number was determined utilizing bead measurement (Beckman Coulter CC Size Standard L10), and the absolute cell numbers were calculated according to the manufacturer’s instructions.

Blood was taken from participants before, immediately postexercise and 1 h post exercise bout by a trained phlebotomist using venepuncture at each timepoint. Peripheral blood was drawn into 1 × 6 mL vacutainers spray‐coated with EDTA anti‐coagulant, and 1 × 6 mL serum gel vacutainer (BD Biosciences, UK), with the first 3 mL of peripheral blood discarded. Total blood differential leukocyte counts were determined using an automated hematology analyzer (XS 1000i, Sysmex, UK). Peripheral blood mononuclear cells (PBMC) were isolated using density gradient centrifugation as described elsewhere (Ross et al. 2016). PBMCs were stained with anti‐CD3‐FITC, anti‐CD31‐APC, and anti‐CD28‐PE‐Cy5 antibodies (all BD Biosciences, UK), with subanalysis of T‐cell subsets using anti‐CD4‐PE (Miltenyi‐Biotec, Germany) and anti‐CD8‐PE (BD Biosciences, UK) antibodies to determine CD4⁺ and CD8⁺ T_ANG cells. PBMCs incubated for 30 min prior to enumeration by flow cytometry.
Peripheral blood collected in the serum gel vacutainer was allowed to clot for 30 min prior to centrifugation (1500g for 10 min at 21°C). Serum was obtained and stored at −80°C for analysis of cytomegalovirus (CMV) serostatus.