Red blood cell lysis buffer

After therapy, the mice were euthanized to obtain tumor tissue. Obtain a single-cell suspension by grinding the tumor and filtering. After being treated with Red Blood Cell Lysis Buffer (Solarbio), the single cells were then collected and washed with PBS and stained with corresponding antibodies following the standard protocol. Following staining, cells were washed, fixed, and then analyzed by flow cytometer.

Fresh venous blood was drawn and leukocytes were isolated within 6 h after blood collection to ensure their survival. Density gradient centrifugation was employed for leukocyte isolation at 18–20 °C using Red Blood Cell Lysis Buffer (Solarbio, Beijing, China), according to the manufacturer’s instructions.

Female C57BL/6 mice (6-8 weeks old) were anaesthetized with pentobarbital and transcardially perfused with cold PBS. Spleens were removed and gently pressed through a 70-μm nylon mesh. After centrifugation at 300 × g for 5 min, cells were treated with red blood cell lysis buffer (Solarbio, Beijing, China) for 3 min on ice. After washing twice with cold PBS, the cells were suspended in complete RPMI 1640 medium supplemented with 10% FBS and 1% GlutaMAX and cultured at a concentration of 2 × 10⁶ cells/ml per well in 24-well plates or 5 × 10⁶ cells/ml per well in 96-well plates at 37°C and 5% CO₂. To induce lymphocyte proliferation, cells were stimulated with ConA (5 μg/ml) or LPS (1 μg/ml). The study was approved by the Animal Ethics Committee of the Third Affiliated Hospital of Sun Yat-sen University ([2019]02-342-01).

mROS was measured using the MitoSOX™ Red mitochondrial superoxide indicator (M36008, Thermo Fisher, USA). MitoSOX is a novel fluorogenic probe for sensitive selective detection of superoxide (instead of reactive nitrogen species) in the mitochondria of live cells. Once in the mitochondria, the MitoSOX reagent is oxidized by superoxide and exhibits red fluorescence. The left lung tissues after modeling from different groups were immediately cut and digested with collagenase type I (17018029, Thermo Fisher, USA) on a shaker at 37°C for 30 min. The samples were filtered two times using a cell mesh, and then the red blood cells were lysed with red blood cell lysis buffer (R1010, Solarbio, China), and followed by centrifugation to obtain the single cells. The obtained mouse single cells or RAW264.7 cells after modeling in vitro were seeded into 24-well plates at a density of 5 × 10⁵ cells/mL. After cells had adhered to the wells, the cells were incubated with MitoSOX (5 μm) for 10 min in the dark and nuclei were visualized by staining with Hoechst 3342 (C0030, Solarbio, China) for 20 min at room temperature as previously described [35 (link)]. The stained cells were then washed with DMEM and observed using a fluorescence microscope (EVOS FL AutoLife Technologies).

Whole blood samples from 126 healthy control, 146 pre-transplant recipients, 30 recipients with stable renal function (STA), and 12 recipients with AR were obtained from the First Affiliated Hospital, College of Medicine, Zhejiang University. The research protocol was approved by the Research Ethics Committee. White blood cell was purified using Red Blood Cell Lysis Buffer (Solarbio, R1010) twice and incubated with conjugated antibiotics including CD4-APC, CD8-PE-Cy™7, CD279 (PD1)-PE, CD57-FITC (BD bioscience, 663498, 335822, 560795, 663497) for 20 minutes at room temperature in dark. After washing, cells were resuspended in PBS. Data was acquired on FACSCanto II flow cytometer (BD Bioscience) and analyzed with FlowJo V10.

Following resection, a representative tumor fragment was isolated and transferred rapidly to the laboratory for study as previously described [20 (link)]. Briefly, tissue was initially cut into segments and subjected to digestion by collagenase type I/II (Thermo Fisher Scientific, USA) and DNAse I (Sigma, USA). The digested pieces were triturated with a 1 ml syringe plunger and passed through a 70 μm cell strainer (Coring, USA). After resuspending in red blood cell lysis buffer (Solarbio, China), live cells were enriched using a Dead Cell Removal kit (Miltenyi Biotec, Germany) as per manufacturer’s instructions. Enriched live cells were washed and counted using a hemocytometer with trypan blue. Cells were then resuspended in PBS containing 0.04% BSA at a concentration of 1 × 10⁶ cells/ml with a viability of > 80% as determined with the Countess. Overall, the entire dissociation procedure took about 2 h from obtaining sample to generating single-cell suspension. The single-cell suspension was then run on the Chromium 10X device (10 × Genomics, USA).
Single-cell library preparation was carried out using Chromium Single cell 3’ Reagent v2 Kits (10 × Genomics, USA) according to the manufacturer’s protocol. Then the library was sequenced on the HiSeq X Ten instruments (Illumina, USA) and 150 bp paired-end reads were generated.

The blood samples (approximately 300 μL) were collected from mouse eyeball using a heparin sodium anticoagulant tube. The triple volume of red blood cell lysis buffer (Solarbio, Beijing, China) was added into the blood for 10 min and then centrifuged at 400 × g for 15 min. The supernatant was discarded, and the sedimentary leukomonocytes were resuspended with 300 μL PBS twice. Subsequently, 1 μL FITC-conjugated anti-CD4 antibody (BD, US) and 2 μL PE-conjugated anti-CD8 antibody (BD, US) were incubated with leukomonocytes for 30 min in a dark box. Then, leukomonocytes were rinsed with PBS 3 times. A total of 10,000 cells were counted for calculating the percentages of CD4 and CD8 positive cells using flow cytometry (CytoFLEX S, Beckman Coulter, US).