Cd90 1 clone ox 7

For the immunophenotypic analysis, third passage MSCc and MSCd were dissociated
and suspended in blocking solution containing cold PBS supplemented with 0.5%
bovine serum albumin (BSA). The cells were treated with rat Fc block (CD32,
Cat#550271; BD Biosciences, San Jose, CA, USA) for 20 minutes before incubation
with antibodies. The following antibodies conjugated with fluorescein
isothiocyanate (FITC), phycoerythrin (PE), or biotin were used: CD29 (clone
Ha2/5, Cat#555005, dilution 1:50; BD Biosciences), CD90-1 (clone OX-7,
Cat#551401, dilution 1:25; BD Biosciences), CD45 (clone 30-F11, Cat#553077,
dilution 1:50; BD Biosciences), and CD34 (clone RAM34, Cat#551387, dilution
1:50; BD Biosciences). The corresponding isotypes (BD Biosciences) were used as
nonspecific binding controls. After incubation at 4ºC for 20 minutes, the cells
were washed with PBS/0.5% BSA, centrifuged at 300 xg for 5 minutes, and
suspended in PBS for data acquisition. DAPI 0.1 µg/mL (Cat# D9452,
Sigma-Aldrich) was added to exclude dead cells. The samples were acquired in a
BD FACSAria II flow cytometer (BD Biosciences) and the resulting data were
analyzed using the software FlowJo, version 10.1.

Tumor tissue was mechanically dissociated and digested in 0.7 mg/mL Collagenase D (Roche) and 3 mg/mL DNase I (Roche) for 30–45 min to obtain single-cell suspension. For experiments looking at tumor-infiltrating leukocytes (TIL), TIL were isolated using Percoll gradient prior to staining. For experiments looking at B16RFP⁺ tumor cells, cells were stained immediately after digestion for 20 min. Cells were blocked with anti-CD16/32 (clone 93; Biolegend) prior to surface staining. Antibodies against the following antigens were used: CD11a (clone M17/4; ThermoFisher), CD8a (clone 53-6.7; BD Biosciences or Biolegend or eBioscience), CD45 (clone 30-F11; Invitrogen or eBioscience), CD45.1 (clone A20; Biolegend), CD3 (clone eBio500A2; eBioscience), CD45.2 (clone 104; eBioscience), CD90.1 (clone OX-7; BD Biosciences), CD127 (clone A7R34; eBioscience or clone SB/199; Biolegend), KLRG1 (clone 2F1/KLRG1; Biolegend or eBioscience), PD-1 (clone RMP1-30; eBioscience), NKG2A/C/E (clone 20d5; eBioscience), LAG3 (clone C9B7W; Biolegend), CD44 (clone IM7; eBioscience or Biolegend or BD Biosciences), Ly6C (clone HK1.4; Biolegend), Ly6G (clone 1A8; Biolegend), CD11b (M1/70; eBioscience or Biolegend), Qa-1^b (clone 6A8.6F10.1A6; Miltenyi Biotec), and PD-L1 (10F.9G2; Biolegend).