Anti cd3 pc5

EDTA-treated whole blood was incubated using the corresponding monoclonal antibodies: anti-CD3-PC5.5 and anti-CD4-APC-A750 (all from Beckman Coulter, Miami, FL, USA); anti-TCRγδ-PE, anti-NKG2D-PCy7 and anti-CD8-APC (all from BDBiosciences, Franklin Lakes, NJ, USA); and anti-CD56-FITC and anti-CD161-FITC (all from BioLegend, San Diego, CA, USA). Innate subsets were analyzed by flow cytometry using a Dx-Flex Cytometer and Kaluza Software (Beckman Coulter, Miami, FL, USA).
NK and NKT cells were considered CD3-CD56+ and CD3 + CD56+, respectively, gated from lymphocytes. MAIT cells were considered Vα7.2+ and CD161+ gated from CD3 + αβTCR+ T cells. MAIT cells expressing CD4 and CD8 were gated from CD3 + CD4+ or CD8+ lymphocytes, respectively, expressing both Vα7.2 and CD161.

Fresh single cells were harvested from passage 5, 12, 20 and resuspended in PBS. Cells were subsequently stained with mouse antibodies anti-CD3-PC5.5, anti-CD3-FITC, anti-CD19-ECD, anti-CD20-APC, anti-CD25-PC5.5, anti-CD45-PC7, anti-CD56-PE, (Beckman Coulter, Brea, CA, United States) according to the manufacturer’s standard procedures. Measurement was performed using a Beckman Coulter Navios flow cytometer.

Based on hsTnI levels, we divided COVID-19 patients into hsTnI-L and hsTnI-H groups, and healthy physical examiners were selected as the control group. Lymphocyte subsets were compared among the three groups.
Peripheral blood samples were collected in ethylenediaminetetraacetic acid K2 (EDTA-K2) collection tubes for lymphocyte subset and hemoglobin A1c detection. Antibodies, including anti-CD3-PC5, anti-CD4-RD1, anti-CD8-ECD, anti-CD45-FITC, anti-CD56-RD1, and anti-CD19-ECD, were purchased from Beckman Coulter (USA). CD3⁺CD4⁺ cells represent T helper cells, CD3+CD8+ cells represent T suppressor cells, and CD4⁺CD25⁺CD127^Dim cells represent T regulatory cells, respectively. Lymphocyte subsets were detected using a flow cytometer (FC500MCL; Beckman Coulter, USA). Hemoglobin A1c was detected using a glycated hemoglobin analyzer (Premier Hb9210, USA).

The internalization of FITC labelled dendrimers was confirmed by flow cytometry. Briefly, 1 × 10⁶ PBMCs or 1.5 × 10⁴ U87MG-CD4⁺CCR5⁺ cells/well were seeded in 24-well plates and treated with the fluorescent dendrimers for 1, 2 or 6 h at 37ºC. After incubation, adherent cells were removed by trypsinization and rinsed with PBS 3% BSA. PBMCs were incubated with anti-CD3-PC5 (Beckman Coulter, Brea, CA, USA) for 30 min at RT and then rinsed with PBS 3% BSA. Viability was assessed in both cell lines with 7-Aminoactinomycin D (7-AAD) (Sigma, St Louis, MO, USA) following manufacturer’s instructions in both cell lines. Lastly, cells were fixed with 4% PFA. Measurements were analyzed using Kaluza software (Beckman Coulter, Brea, CA, USA).