Anti human cd45

Human immune cell reconstitution in hu-mice was analyzed by flow cytometry (FCM) using various combinations of the following monoclonal antibodies: anti-human CD45, CD3, CD4, CD8, CD45RA, CD45RO, CD69, CCR7, CD31 (all purchased from Biolegend, San Diego, CA, USA); and anti-mouse CD45 (BD Pharmingen) and Ter119 (Biolegend, San Diego, CA, USA). Peripheral blood was collected from tail vein into heparinized tubes, and mononuclear cells were purified by density gradient centrifugation with Histopaque-1077 (Sigma-Aldrich, St. Louis, MO, USA). FCM analysis was performed on a FACS Fortessa (BD Biosciences). Dead cells were excluded from the analysis by gating out lower forward scatter and high propidium iodide–retaining cells. Data analysis was performed using FlowJo 10.3 software.

The expression of surface markers on MSCs was
investigated using the following antibodies. Anti-mouse
CD34 (PE, eBiosience, USA), anti-mouse CD44 (APC,
BD, USA), anti-mouse CD45 (APC-cy7, Biolegend,
USA), anti-mouse CD73 (PE, BD, USA), anti-mouse
CD90 (APC, BD, USA), anti-mouse CD105 (PE,
eBiosience, USA), anti-mouse Sca1 (FITC, Biolegend,
USA), anti-mouse CD3 (PE, BD, USA), anti-human
CD90 (APC, Biolegend, USA), anti-human CD105 (APC,
Biolegend, USA), anti-human CD29 (PE, eBiosience,
USA), anti-human CD45 (FITC, Biolegend, USA) and
anti-human CD34 (PE, eBiosience, USA). Passage 2 cells
were used for the analysis of cell surface markers by flow
cytometry (FACS calibur, Becton Dickinson, USA). For
flow cytometry analysis, 10,000 events were counted and
data were analyzed using the flowJo software.

We plated 10,000 OVCAR3 cells per well in a 96-well plate and, after 18 hours, added primary PBMCs. After a 4-hour coincubation, we stained the cells with 7AAD (Invitrogen), annexin V (BioLegend), and anti–human CD45 (304002, BioLegend), then performed a flow cytometry–based cytotoxicity assay as described previously (7 (link)). We stably transfected K562 and K562-FSHR with firefly luciferase. We plated 20,000 K562 and K562-FSHR cells expressing luciferase in a 96-well plate and coincubated them for 5 hours with PBMCs. After the incubation, we lysed the cells and measured luciferase expression using CytoTox Glo (Promega) as previously described (32 (link)). Cytotoxicity was calculated as (maximum viability control – individual well)/(maximum viability control – maximum death control) × 100 as a percentage or relative to the control (PBMCs with mouse IgG2a isotype control C1.18.4).

Cells suspended in 100 μL of PBS were incubated with antibodies at 4 °C for 30 min. The samples were measured on BD Fortessa and analyzed by FlowJo software (Tree Star). Antibodies used in our study were listed: anti-Human CD34 (BioLegend, Pacific Blue, clone 581), anti-Human CD34 (BioLegend, PE, clone 581), anti-human CD201 (BioLegend, APC, clone RCR-401), anti-Human CD43 (BioLegend, APC, clone 10G7), anti-Human CD45 (BioLegend, FITC, clone HI30), anti-Human CD90 (BD Pharmingen, APC, clone 5E10), and CD24 (BioLegend, PE, clone ML5).

The MACS CD206⁺ enriched population of lung resident macrophages were incubated with FcR Block (422302; BioLegend) for 5 min and stained at a dilution of 1:50 with one of two panels of directly conjugated antibodies for 30 min at 4°C: anti-human CD45 (563792; BD), CD204 (371906; BioLegend), CD206 (321132; BioLegend), CD14 (562698; BD Biosciences), CD16 (302028; BioLegend), ACE2 (FAB933P; R&D), HLA-DR (307618; BioLegend), CD11b (393114; BioLegend), CD11c (301644; BioLegend); anti-human CD45 (324016; BioLegend), CD204 (371904; BioLegend), and CD206 (321103; BioLegend). Stained cells were then washed with FACS buffer (2% FBS in PBS) three times, and then incubated with cell viability marker propidium iodide (PI, 1 μg/ml, 421301; BioLegend). Flow cytometry was performed on a FACS Aria II (BD Biosciences).
Living (PI⁻) single, immune (CD45⁺), and lung resident macrophages (CD206⁺) were stained for the above panel of cell surface antigens that have previously been suggested to segregate them into AMs and IMs, and that were differentially expressed according to the scRNA-seq transcriptomic profiles obtained from lung slice culture and sorted into CD206⁺CD204^hi and CD206⁺CD204^lo populations. The sorted populations were directly subjected to 10x single-cell mRNA sequencing at BSL2 as described above, which confirmed their molecular identities as AMs and IMs, respectively.