Anti cd19 ecd

Fluorescence-activated cell sorting was used to measure cell counts for leukocytes, neutrophils, T cells, CD4+T cells, CD8+T cells, NK cells, and B cells (anti-CD45 FITC; anti-CD56 PE, anti-CD16 PE, anti-CD19 ECD, anti-CD3 PC5, IgG1 PC7, anti-CD4 PE, anti-CD8 PCD, anti-CD3 PC5 [Beckman Coulter]).

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).

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.

Flow cytometry was performed as previously described [26 (link),27 (link)]. Analysis of the expression levels of cell-surface markers was performed by flow cytometry. DCs were blocked for 5 min with PBS containing 10% heat inactivated human AB serum (Sigma-Aldrich, St. Louis, MO, USA), and then stained by adding the antibodies to the same buffer. All other cell types were stained on PBS containing 3% FBS. Cells were labeled with anti-CD1a-PE, anti-CD3-PC7, anti-CD4-FITC, anti-CD14-PC7, anti-CD19-ECD, anti-CD25-PC7, anti-CD69-PC5, anti-CD80-FITC, anti-CD86-PC5.5, anti-CD86-PE, anti-HLA-DR-ECD, anti-HLA-DR-PB, anti-HLA-DR-PE (all from Beckman Coulter Inc., Brea, CA, USA), anti-CD8-Pacific Blue, anti-CD71-APC-Cy7, anti-CD197-APC-Cy7 (CCR7) (all from BioLegend, San Diego, CA, USA), anti-CD83-APC (BD Biosciences, Franklin Lake, NJ, USA), and anti-CD3-PO (Abcore, Ramona, CA, USA).
Levels of surface expression on cells were estimated by flow cytometry (Gallios; Beckman Coulter, Brea, CA, USA) and analyzed using Kaluza software (Beckman Coulter, Brea, CA, USA). Entry of FITC-labeled p24 peptide into the DCs was also estimated by flow cytometry. DCs were collected 2 h, 24 h, and 48 h after complex addition, washed with PBS, and acid washed, in order to eliminate any peptide attached to the cell surface. Fluorescence was measured by flow cytometry.

NK cells and PMN-MDSCs were isolated from PBMCs of mobilized and non-mobilized donors using NK isolation kit and CD66b⁺ microbeads (purity >98%, data not shown) following manufacturer’s instruction (Miltenyi Biotec, Bergisch Gladbach, Germany). Before starting any experiment, we determined the purity of isolated cells by flow cytometry using anti-CD3-APC, anti-CD19-ECD, and CD56-PC7 (Beckman Coulter, Brea, CA) for NK cells. PMN-MDSCs were labeled with anti-CD3-AF700, anti-CD19-AF700, anti-CD11b-FITC, anti-CD33-PC7, anti-HLA-DR-PE, anti-CD14-ECD, anti-CD45-KrOr, and anti-CD66b-APC desiccated in the Duraclone custom design platform (Beckman Coulter, Brea, CA) adding anti-CD56-BV650 (BioLegend, San Diego, CA) and following manufacturer’s instruction. After the staining procedures, cells were acquired at Cytoflex LX and analyzed with Cytexpert software (v2.4, Beckman Coulter, Brea, CA). Freshly isolated NK cells were immediately used for functional studies and gene expression evaluation.