Id7000

To analyze tumor-infiltrating immune cells, subcutaneously tumors were dissected and transferred into RPMI 1640 medium, disrupted mechanically with scissors, digested for 1 h at 200 rpm/min using a mouse tumor dissociation kit at 37°C, and dispersed through a 100 μm cell strainer to remove residual tissue (BD Biosciences). Single cells were washed and stained with antibodies for 30 min at room temperature. Dead cells were excluded by staining with the Zombie fixable viability kit for 30 min (BioLegend). Fluorescence data were acquired on a Sony ID7000 (Sony) and analyzed using FlowJo software. Tumor-infiltrating cells isolated from tumor tissues, T cells derived from the spleen, or BMDMs were processed for surface labeling with antibodies against CD45, CD11b, F4/80, CX3CR1, CD3, CD8, PD1, TIGIT, TIM3, CD206, and I^AI^E. The antibodies used for flow cytometry were purchased from Biolegend. These antibodies were diluted according to the manufacturer’s instructions (1:1,000).

Cell surface expression and activation of β1-Integrin was assessed by FACS (Sony ID7000), as described previously [31 (link)]. Cells were detached using trypsin, washed, and resuspended for staining with PE-conjugated mouse anti-human CD-29 antibody or FITC-conjugated HUTS-4 antibody. Data were analyzed using FlowJo software.

Mouse BM and PB samples were prepared as we described [8 (link), 24 (link)]. To analyze the phenotype of normal hematopoietic cells, samples were stained with anti-lineage cocktail (anti-CD3e, anti-CD11b, anti-Gr-1, anti-B220 and anti-Ter-119), anti-Sca-1, anti-c-Kit, anti-CD34, anti-Flk2, anti-CD150, anti-CD48, anti-CD127, anti-CD16/32, anti-Gr-1, anti-CD11b, anti-B220, anti-CD3e, anti-CD45.1 and anti-CD45.2. To analyze the phenotype of LSCs, anti-lineage cocktail, anti-Sca-1, anti-c-Kit, anti-CD34 and anti-CD16/32 were used. The apoptosis, cell cycle, in vivo BrdU incorporation and intracellular staining assays were performed following our previous protocols [24 (link)]. Flow cytometry analysis was conducted using a FACSVerse (BD Biosciences, San Jose, CA, USA) or ID7000 (Sony Biotechnology, Tokyo, Japan). For cell sorting, a Direct Lineage Cell Depletion Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) was used to delete the mature cells and then samples were stained with above antibodies, followed by sorting using a FACSAria III (BD Biosciences). Gating strategies are referred to our previous studies [8 (link), 24 (link)]. The details of antibodies are provided in Supplementary Table S1.

Mice were sacrificed to prepare single-cell suspensions of kidney cells after IRI. After removing red blood cells, cells were wash with PBS. Zombie was used to distinguish living cells. After blocking with CD16/32, cell suspensions are incubated with primary antibodies. Incubate for 15 min at a concentration of 1:100 at 4°C from light. Flow cytometric analysis were analyzed using a ID7000 (Sony, Japan) flow cytometer. Primary macrophage isolation was labelled and separated using the same antibodies. Data analysis was conducted by Flow Jo software (Treestar Inc., United States). The antibody used in this study is listed in Supplementary Table S1.

The following antibodies from BioLegend were diluted in flow cytometry buffer (DPBS, 2% FBS, 2 mM EDTA): PerCP-Cy5.5-conjugated anti-human CD2 (RPA-2.10, 0.25 μg ml⁻¹, #300216), PE-Cy7-conjugated anti-human CD3e (UCHT1, 0.75 μg ml⁻¹, #300420), Pacific Blue or PE-Cy7-conjugated anti-human CD20 (2H7, 1 μg ml⁻¹, #302320 or #302312), APC-Cy7 or PE-Cy7-conjugated anti-human CD69 (FN50, 0.5 μg ml⁻¹, #310914 or #310912), and APC anti-HA.11 epitope tag (16B12, 0.5 μg ml⁻¹, #901524). BV395-conjugated CD20 (2H7, 0.25 μg ml⁻¹, #563782) was purchased from BDbiosciences. Propidium Iodide Ready Flow Reagent (Thermo Fisher, #R37169) was used as viability dye. GFP and BFP were additionally analyzed to identify TCR activation and minigene translation efficiency. Cells were washed before staining for 20 min at 4°C in the dark. Samples were then washed and analyzed using a BD FACSAria Fusion or BD FACSAria II cell sorter, or in high throughput using a Sony ID7000 spectral analyzer. Data analysis was performed using FlowJo (10.6.1). For single-variable analysis, the percentage of positive cells was calculated as the integrated area under the histogram not overlapping with control samples using FlowJo’s population comparison (Overton) method.