Anti cd45 apc cy7

Sputum cells were blocked with anti-CD16/CD32 antibodies (BioLegend, USA) for 10 min and then stained with Live_Dead (Zomnie Aqua, BioLegend) for 20 min at 4 °C. Human induced sputum cells were then stained with the following antibodies: anti-CD45 (APC/Cy7; BioLegend), anti-CD11c (APC; BioLegend), anti-CD68 (PE; Invitrogen, CN), anti-CD86 (PE/Dazzle594; BioLegend), anti-CD16 (Percp/Cy5.5; BioLegend), anti-CD11b (PE/Cy7; BioLegend), and anti-CD15 (FITC; BioLegend). Flow cytometry was performed with a Cytek Dxp Athena flow cytometer, and the data were analyzed by using FlowJo (version 10) software.

Briefly, the fresh tissue samples from two human PC tissues and seven mouse-derived allografts of KPC1199 cells were mechanically chopped and digested by collagenase IV at 37 °C for 30 min. The digested suspension was combined with DNase at room temperature for 5 min, washed twice with phosphate-buffered saline buffer containing 2% serum, and then filtered through the 100 μm filter. We used markers of CD29, PDPN, EpCAM, and CD45 to separate tumor epithelial cells (CD45⁻CD29⁻ or PDPN⁻EpCAM⁺), CAFs (EpCAM⁻CD45⁻CD29⁻ or PDPN⁺), and tissue leukocytes (CD29⁻ or PDPN⁻EpCAM⁻CD45⁺) in human and mouse tumor specimens, respectively. The digested single cells were washed twice and centrifuged for 5 min at 500× g, and then 1 μg/mL of antibody was added. Then, the samples were kept at 4 °C for 30 min in a dark place. Flow cytometry was employed using a BD Flow Cytometry Analysis Celesta cell sorter (Becton Dickinson, New York, NY, USA). The side-scatter width versus side-scatter region and the forward-scatter width versus forward-scatter height were applied to remove dead cells and cell clumps. Antibodies including anti-EpCAM-PerCR/Cy5.5 (BioLegend, San Diego, CA, USA, #324214), anti-CD45-APC-Cy7 (BioLegend, #368515), and anti-CD29-Alexa Fluor^® 488 (BioLegend, # 303015) were verified according to the manufacturer’s website.