Anti cd45 percp

24 hours after wounding, mice were sacrificed and eyes enucleated. Dissected corneas were cut into quarters and digested in Collagenase Type 1 (Sigma) 820 U/mL in DMEM/F12 + 10% fetal bovine serum at 37°C for 1 hour, vortexing every 20 minutes. Digests were triturated until tissue was completely broken up, and digestion continued for an additional 20 minutes. Cell suspensions were filtered through a 70 μm nylon mesh and pelleted at 340 x g for 10 minutes. Cells were stained in 50 μl staining buffer (phosphate buffered saline, 1% fetal bovine serum) by addition of Fc-Block, anti-CD45-PerCP, anti-Gr1-PE (clone RB6-8C5), and anti-CD11B-AF647 (all BD Biosciences) at 1:50 for an incubation of 30 minutes on ice in the dark. Stained cells pooled from 6 corneas were washed by centrifugation from staining buffer and fixed in 300 ul 1% paraformaldehyde in PBS before analysis by flow cytometry on a FACS Aria III Flow Cytometer (BD Biosciences). For determining cell counts in individual corneas, each sample was spiked with 20,000 fluorescent counting beads (CountBright absolute counting beads; Invitrogen). and the bead count was used to calculate absolute numbers of immune cells per cornea[31 (link)].

Cells from lamina propria and lungs were stimulated with PMA (Sigma-Aldrich) 50 ng/ml, Ionomycin (Sigma-Aldrich) 500 ng/ml and Brefeldin A (Biolegend, San Diego, CA, USA) 5 µg/ml for 4 h at 37 °C and 5% CO₂. T-cell surface staining was performed for 30 min at 4 °C using the following antibodies diluted in PBS: anti-CD45 PercP (BD, Franklin Lakes, NJ, USA), anti-CD4 APCCy7 (Biolegend) and anti-CD8 FITC (Biolegend). Cells were then fixed and permeabilised using the Cytofix/Cytoperm kit (BD). Intracellular staining was performed for 30 min at 4 °C with the following antibodies diluted in Perm/wash buffer (BD): anti-CD3 APC (BD), anti-IL-9 PE (Biolegend) and anti-IFN-γ PECy7 (Biolegend). Lung cells were also stained for mast cell detection with anti-CD45 PercP and anti-CD117 APC (Biolegend). Samples were acquired using a FACS Canto II (BD) and analysed using FlowJo software (version 9.0.2, Tree Star).

Immunofluorescence staining of monocyte subsets and their co-stimulatory molecules were performed by using fluorochrome-conjugated monoclonal antibodies (mAbs) consisted of anti-CD3-fluorescein isothiocyanate (FITC) (clone SK7, T-cell marker); anti-CD19-PE-Texas Red (clone SJ25-C1); anti-CD86-PE/Dazzle594 (clone IT2.2); anti-CD45-peridinin chlorophyll protein (PerCP) (clone 2D1); anti-CD275(ICOSL)-allophycocyanine (APC) (clone 2D3); anti-CD3-allophycocyanin/cyanine 7 (APC-Cy7) (clone SK7); anti-CD19-APC-Cy7 (clone HIB19); anti-CD56-APC-Cy7 (clone HCD56); anti-CD40-Pacific Blue (clone 5C3); anti-CD16-BV510 (clone 3G8); anti-CD14-BV570 (clone M5E2) and anti-CD80-BV605 (clone 2D10). Human IgG isotype control antibodies conjugated with APC, Pacific Blue, PE/Dazzle594 and BV605 were used for each specific co-stimulatory molecules. Fluorochrome-conjugated mAbs were purchased from Biolegend (San Diego, CA, USA), except anti-CD3-FITC and anti-CD45-PerCP that were purchased from BD Biosciences (San Jose, CA, USA) and anti-CD19-PE-Texas Red from Southern Biotech (Birmingham, AL, USA).

BMMCs and BMA were analyzed for the expression of cell-surface antigens with direct three-color analysis using fluorescein isothiocyanate (FITC)-conjugated, phycoerythrin (PE)-conjugated and peridinin chlorophyll protein complex (PerCP)-conjugated monoclonal antibodies as previously reported [33 (link), 34 (link)]. The following antibodies were used for analysis: immunoglobulin G1 control PE (eBioscience, San Diego, CA, USA), immunoglobulin G1 control PerCP (Becton Dickinson, San Jose, CA, USA), immunoglobulin G1 control FITC (BD Bioscience, Franklin Lakes, NJ, USA), anti-CD34-FITC (BD Bioscience), anti-CD34-PerCP (Becton Dickinson), anti-KDR-PE (R&D Systems, Minneapolis, MN, USA), anti CD45-PerCP (BD Bioscience), and anti-CD133–PE (Miltenyi Biotec, Bergisch Gladbach, Germany). The quantification of total mononuclear cells in BMMCs and BMA was determined by Turk’s solution. For FACS analysis, 5 × 10⁵ events were acquired and scored with a FACSCalibur analyzer (Becton Dickinson). Data were processed using the Macintosh CELLQuest software program (Becton Dickinson).

Flow cytometry was performed using a FACSCalibur Cytometer (BD Biosciences); a phenotyping kit (MSC phenotyping kit, Miltenyi) was used to characterize the tMSCs. The following antibodies were used: anti-CD34PerCP, anti-CD45PerCP, anti-CD20PerCP, anti-CD14PerCP, anti-CD73APC, anti-CD9FITC, and anti-CD105PE (BD Pharmingen). Matched isotype controls were applied to determine background fluorescence levels.

Samples of EDTA anticoagulated peripheral blood (2 mL) were collected from patients with the Delta variant on admission. All the samples were tested within 6 h. Briefly, CD3+/CD4+/CD8 + T-cell, CD19 + B-cell, and CD16 + CD56 + NK-cell counts (cells/µL) were measured by multiple-color flow cytometry with human monoclonal anti-CD3-fluorescein isothiocyanate (FITC), anti-CD8-phycoerythrin (PE), anti-CD45-PerCP, anti-CD4-allophycocyanin (APC), anti-CD16-PE, anti-CD56-PE, and anti-CD19-APC antibodies (BD Multitest) according to the manufacturer’s instructions. The cells were analyzed on a BriCyte E6 flow cytometry system (Mindray).