Pe positive selection kit

CD34+ progenitor cells were isolated and enriched from cord blood as reported previously.(51) MK cultures of 10⁵ cells/ml were supplemented with 100 ng/ml thrombopoietin (rhTPO CellGenix, Freiburg, Germany) and 10 ng/ml IL1b (R&D, Minneapolis, MN, USA) in CellGro media (CellGenix, Freiburg, Germany). Media and cytokines were rejuvenated at days 3 and 6. At day 10, mature MKs were immunopurified using an anti-CD42b PE conjugated antibody (Pab5, NHS Blood and Transplant, IBGRL, Bristol, England) and a PE positive selection kit (STEMCELL Technologies, Vancouver, Canada). MK purity was verified by FACS and shown to be >95% for CD41a (BD, San Jose, CA, USA) and CD42b (IBGRL, Bristol, England).

Stromal vascular fractions (SVF) of epididymal fat were isolated by collagenase digestion as previously detailed (21 (link)). In brief, minced adipose tissue was placed in Hank's Balanced Salt Solution containing 2% bovine serum albumin (BSA) and 1 mg/mL collagenase then homogenized with a gentleMACs dissociator (Miltenyi Biotec), and incubated for 30–40 min at 37°C with gentle shaking until fully dissociated. SVF cells were pelleted and floating adipocytes removed. SVFs were filtered, RBC-lysed, and washed, then used immediately for either flow cytometry or ATM isolation. To isolate ATMs, SVF cells were labeled with either Fluorescein isothiocyanate (FITC)- or Phycoerythrin (PE)-anti-F4/80 antibody (Clone: BM8, Biolegend) then F4/80+ ATMs were immunomagnetically selected with either FITC- or PE-Positive Selection Kit (StemCell Tech). Flow cytometry was used to evaluate selection purity, which was routinely >75%.

For isolation of CD4⁺ T cells, single-cell suspensions of splenocytes from 4T1 tumor-bearing control mice (day 16 after inoculation) were isolated by an Easysep CD4⁺ T cell negative selection kit (StemCell Technologies, Vancouver, BC, Canada). For isolation of MDSCs, single-cell suspensions of splenocytes from indicated groups were firstly incubated with Gr1-PE (listed in Supplementary Table 1) and then isolated with a PE positive selection kit (StemCell Technologies, Vancouver, BC, Canada).

Normal lung tissues were digested with 2 mg ml^-1 collagenase type I (Worthington Biochemical), 2 mg ml^-1 collagenase type IV (Worthington Biochemical), and 0.2 mg ml^-1 DNase I (Sigma-Aldrich) at 37 °C for 1 h, and terminated with culture medium containing 10% FBS. The single-cell suspension was obtained by filtering through a 70 μm cell strainer. CD31-positive primary ECs were isolated using anti-mouse CD31-PE antibodies (Biolegend, San Diego, CA, USA) and PE positive selection kit (StemCell, Vancouver, BC, Canada), and cultured in endothelial cell culture medium (Procell, Wuhan, China) for 5 days before use.
After treatment with 2 μg ml^-1 IGFBP7 and/or 10 μg ml^-1 M057 for 24 h, mouse primary ECs were seeded (1 × 10⁴ cells/well) in an ibidi plate precoated with endothelial growth factor-reduced Matrigel (BD Bioscience, Sab Jose, CA, USA). Mouse primary ECs were stained with 6.25 μg/ml calcein AM (PromoKine, Heidelberg, Baden‐Württemberg, Germany) for 15 min. Images for the capillary tubes were captured under an inverted fluorescence microscope and analyzed by ImageJ (NIH) angiogenesis analyzer plugins.

CD3⁺ T cells were purified from splenocytes, as described previously [71 (link)]. In brief, the splenocytes were collected from mice and labeled with PE-conjugated anti-CD3 antibody (BioLegend, Clone:17A2). Immunomagnetic selection was achieved by the use of PE Positive Selection Kit (STEMCELL Technologies; Cambridge, MA, USA).

For isolation of CD4⁺ and CD8⁺ T cells, spleens from the treated mice and 4T1 tumor-bearing control mice were harvested on day 21 after treatment, and single cell suspensions were prepared using a GentleMACS dissociator (Miltenyi Biotec). CD4⁺ and CD8⁺ T cells were isolated by an Easysep CD4⁺ T cell negative selection kit and an Easysep CD8⁺ T cell negative selection kit (StemCell Technologies, Vancouver, BC, Canada), respectively. CD4⁺ and CD8⁺ T cells with a purity of >90% were used for experiments. For isolation of Tregs, the isolated CD4⁺ T cells were then incubated with CD25-PE (clone 3C7, Biolegend, USA) and isolated with a PE positive selection kit (StemCell Technologies, Canada). The isolated cells were then verified by flow cytometry, and more than 85% of cells expressed Foxp3.