Pe conjugated anti cd31

Cells were detached using 0.2% EDTA/PBS. For VE-cadherin and CD31 staining, cells were blocked with 2% bovine serum albumins (BSA) (019–23293, Wako, Osaka, Japan)/PBS for 30 minutes. For αSMA and collagen type I staining, the cells were fixed in 4% paraformaldehyde (09154–85, Nacalai) for 10 minutes at room temperature, and blocked/permeabilized with 2% BSA/0.1% Triton X-100/PBS for 30 minutes. The cells were incubated with primary and subsequently secondary antibodies in 2% BSA/PBS for 1 hour at 4°C. Samples were analyzed with Guava easyCyte (Millipore, Billerica, MA, USA). Antibodies used were Cy3-conjugated anti-αSMA (1:500; C6198, Sigma), PE-conjugated anti-CD31 (1:50; 303105, Biolegend, San Diego, CA, USA), anti-collagen type I (1:80; AB758, Millipore) and Alexa Fluor 647-conjugated anti-goat IgG (1:200; A-21447, Thermo Fisher Scientific), and Alexa Fluor 647-conjugated anti-VE-cadherin (1:50; 561567, BD Biosciences, San Jose, CA, USA).

Mouse kidney tissue was diced and digested with Collagenase-I at 37°C for 1 hour. The suspension was filtered through a 40 um strainer to remove cell pellets and washed in PBS to generate a single cell suspension after lysis of red blood cells (BD, USA). The cells were washed twice with PBS. The third generation of hAD-MSCs were trypsinized and washed twice with PBS. The cells were then incubated with the following fluorescent antibodies and corresponding isotype controls (Cat.Number: 400605, 400611 and 400507, Biolegend, USA) for 30 minutes shielded from light at room temperature: FITC-conjugated anti-CD45, APC-conjugated anti-CD11b, PE-Cy7-conjugated anti-CD3, PE-conjugated anti-F4/80, APC-conjugated anti-CD34, PE-conjugated anti-CD31, FITC-conjugated anti-CD90, APC-conjugated anti-CD44 and PE-conjugated anti-105 (Biolegend, USA). The positive cells were sorted using a BD FACS and the data were analysed using the FlowJo v7.6.3 software.

4-NQO-induced esophagus SCC were dissected, minced and digested in 2 mg/mL of collagenase I (A. G. Scientific, San Diego, CA, USA, C-2823) for 1 h. Collagenase I activity was blocked by the addition of 5 mM EDTA and incubation for 15 min. Trypsin (0.125%) (Capricorn Scientific, Ebsdorfergrund, Germany, TRY-2B10) was then added for 15 min and then the cells were rinsed in PBS supplemented with 2% FBS. All incubations were done on a rocking plate at 37 °C.
Immunostaining was performed on single cell suspension using PE-conjugated anti-CD45 (1:500, BioLegend, San Diego, CA, USA), PE-conjugated anti CD31 (1:500, BioLegend), PE-conjugated anti-CD140a (1:500, BioLegend) and Hoechst 33258 (1:10,000, Molecular Probes/Thermo Fisher Scientific, Waltham, MA, USA, H3569), during 45 min at 4 °C on a rocking plate. Living epithelial cells were selected by forward scatter, side scatter, doublets discrimination and by Hoechst dye exclusion. YFP+/Lin- cells were selected based on the expression of YFP and the exclusion of CD45, CD31, CD140a (Lin-). Control esophageal cells were selected based on the expression of APC-Cy7 EpCam (1:250, BioLegend) and the exclusion of CD45, CD31, CD140a (Lin-). Fluorescence-activated cell sorting analysis was performed using FACSAria III and FACSDiva software (BD Biosciences, San Jose, CA, USA).

Single-cell suspensions from mouse tissues were prepared as previously described [20 (link)]. Tumor tissues were cut in half, and one half was minced and processed for flow cytometry analysis. Cells were stained in PBS/0.5% FBS/2 mM EDTA with the following fluorochrome-conjugated antibodies: BV421-conjugated anti-Ly6C (HK1.4, #128031, 1:100), PerCP-Cy5.5-conjugated anti-CD45 (30-F11, #103132, 1:200), PE-conjugated anti-EPCAM (G8.8, #118205, 1:200), PE-conjugated anti-CD31 (390, #102407, 1:200), PE-Cy7-conjugated anti-PDPN (8.1.1, #127411, 1:100), APC-Cy7-conjugated anti-MHCII (M5/114.15.2, #107628, 1:300) (from BioLegend); FITC-conjugated Hypoxyprobe-1-MAb1 (4.3.11.3, #FITC-Mab, 1:200) (from Hypoxyprobe, Inc). The viability marker Zombie Aqua was purchased from BioLegend (#423102). Flow cytometry was performed on a ZE5 Cell Analyzer (Bio-Rad), and data were analyzed using FlowJo software.

For IVM, Brilliant Violet 421-conjugated anti-Ly6G (neutrophils; clone 1A8, BD Biosciences, San Diego, CA, USA, 1.6 μg), AlexaFluor 647- or PE-conjugated anti-CD49b (platelets; clone HMα2, BioLegend, San Diego, CA, USA, 1.6 μg), PE-conjugated anti-CD31 (endothelium; clone 390, Biolegend, 0.8 μg), goat anti-mouse histone H2Ax (clone M20, Santa Cruz Biotechnology, USA, 2 μg) was conjugated to AlexaFluor 555 and goat–anti-mouse neutrophil elastase (M18, 2 μg) was conjugated to AlexaFluor 647 using a labelling kit as per the manufacturer’s instructions (Life technologies, Carlsbad, CA, USA). Quantities and clones of antibodies used to label neutrophils and platelets have been previously optimized and demonstrated to have minimal impact on cellular recruitment and or clearance through mechanisms such as antibody-dependent cellular cytotoxicity (28 (link), 29 (link)). Thrombin was visualized with 5-FAM/QXL^® 520 FRET substrate (SensoLyte^® 520 Thrombin Activity Assay Kit, AnaSpec, Inc., Fremont, CA, USA). FITC- or AF647-albumin was used to delineate the vasculature.