Anti cd4 phycoerythrin

The following anti-human monoclonal antibodies were used for surface phenotype and intracellular cytokine staining: anti-CD4-fluorescein isothiocyanate (FITC); anti-CD4-phycoerythrin (PE); anti-CD161-PE; anti-CCR6-PE; anti-CD45RO-PE; anti-CD147-peridinin chlorophylla protein cyanine 5.5 dies (Percp–cy5.5); anti-interferon (IFN)-γ-FITC (all from BD Biosciences); and anti-IL-17A-allophycocyanin (APC; eBiosciences). Anti-mouse monoclonal antibodies included: anti-CD4-Percp; anti-IL-17A-APC; and anti-IFN-γ-FITC (all from BD Biosciences). Appropriately conjugated IgG antibodies were used as isotype controls. Cells were acquired on a FACSCalibur flow cytometer (BD Biosciences) and analyzed using Cell Quest software (BD Bioscience) and FlowJo 7.6.1 software (Tree Star).

Lymphocyte subset percentages were analyzed before initial treatment. EDTA anticoagulated peripheral blood (2 ml) were collected from patients with COVID-19 on admission (18 (link)). All samples were tested within 6 h of being obtained. Briefly, CD3+ total T-lymphocytes, CD19+ B-cell, CD3+/CD4+/CD8+ T-cell, and CD16+CD56+ NK-cell counts (cells/μl) were measured by multiple-color flow cytometry with human monoclonal anti-CD19-PE, anti-CD3-fluorescein isothiocyanate (FITC), anti-CD4-phycoerythrin (PE), anti-CD8-allophycocyanin (APC), anti-CD16-APC, and anti-CD56-PE antibodies (BD Multitest) according to the manufacturer's instructions. The cells were analyzed on a Partec Cube 6 Flow cytometry system (Sysmex Europe GmbH, Germany).

Phenotyping of spleen cells was performed using the following antibodies: anti-B220-phycoerythrin, anti-CD4-phycoerythrin, anti-Mac1-PerCPCy5.5 (all BD Biosciences) and anti-CD8-FITC (Serotec). Spleen cell suspensions in PBS + 0.1% BSA (wash buffer) were stained with combinations of antibodies or isotype controls for 30 minutes at 4°C, red cells were lysed using Pharmlyse (BD Biosciences) and cells were analysed using an Accuri C6 flow cytometer and Cflow Sampler software (BD Biosciences). Analysis of activated caspase 3 expression was performed by flow cytometry using the PE Active Caspase 3 Apoptosis Kit (BD Biosciences) according to manufacturer's instructions. Sorting of spleen lymphoid and myeloid cells was performed using a combination of anti-B220-phycoerythrin and anti-CD3-phycoerythrin to identify lymphoid cells and anti-Mac1-PerCPCy5.5 to identify myeloid cells. Sorting of blast cells from primary lymphomas was performed by staining cells with anti-B220-phycoerythin and anti-CD45-FITC; in the B220⁺ population, blast cells were identified using CD45 and SSC as reported in [27 (link)]. All sorting was performed using a FACSAria (BD Biosciences).

Fluorescence-activated cell sorting analysis was performed using a BD LSRFortessa™ (BD Biosciences, San Jose, CA). The following antibodies were used to classify cells: anti-CD19-fluorescein isothiocyanate (FITC) (Miltenyi Biotec, Friedrich Gladbach, Germany), anti-CD3-allophycocyanin (APC), anti-CD4-APC, anti-CD4-phycoerythrin (PE), anti-CD24-peridinin chlorophyll protein complex-Cy 5.5 (PerCP Cy5.5), anti-CD25-APC, anti-CD27-PE, anti-CD39- brilliant violet 421 (BV421), anti-CD45-V500, anti-CD73-APC, anti-IFN-γ-BV421, and anti-IL-10-BV421 (all from BD PharMingen, Franklin Lakes, NJ), anti-CD80-APC, anti-CD86-APC monoclonal antibody (mAb) (Biolegend, San Diego, CA), Annexin V- FITC (BD PharMingen), and DAPI (Cell Biolabs, San Diego, CA). The CD19⁺CD24^hiCD27⁺ B cell subset was sorted using a Moflo XDP cell sorter (Beckman Coulter, Brea, CA) with 90% to 95% purities.