C tube

Peripheral blood mononuclear cells (PBMC) were isolated from PB of patients and healthy donors by density gradient separation using Ficoll-Paque Plus (GE Healthcare). PBMC were then suspended in AIM-V medium (Life Technologies).
To isolate tumor infiltrating lymphocytes (TIL), tumor tissue was cut into small pieces and put into C-tubes (Miltenyi) containing 9.8 ml of AIM-V medium, 100 μl DNAse I (10 mg/ml; Sigma Aldrich), and 100 μl of collagenase/hyaluronidase (3000 IU/ml collagenase and 1000 IU/ml hyaluronidase; Stem Cell). The C-tubes were then processed with the GentleMACS (Miltenyi), according to the manufacturer’s instruction. The resulting suspensions were then filtered through 40 μm cell strainer, washed and then suspended in AIM-V medium. To obtain tissue homogenates for lymphocyte activation assay, small part of tumor tissues were sliced and homogenized in 2X phosphate-buffered saline (PBS) by using Bio-Gen PRO200 Homogenizer (Pro Scientific). The tissue homogenates were then heated to 100°C for five minutes to establish protein denaturation.
Single cell suspensions from SN were obtained by gentle pressure homogenization in AIM-V medium through a 40 μm cell strainer. The single cell suspension were then washed and suspended in AIM-V medium.

Single-cell suspension was prepared as described in Adam et al. (2017) (link) with all the procedures run on ice or in cold room. Mice were euthanized and the lungs were perfused with PBS as described above. The lungs were inflated with cold active protease solution (5 mM CaCl2, 10 mg/mL Bacillus Licheniformis protease), dissected and transferred to a petri dish, and the heart, thymus and trachea were removed. The lobes were minced using a razor blade. The minced tissue was then immersed in extra cold active protease solution for 10 min and triturated using a 1 mL pipet. This Homogenate was transferred to a Miltenyi C-tube with 5mL HBSS/DNase (Hank’s Balanced Salt Buffer) and the Miltenyi gentleMACS lung program was run twice. Subsequently, this suspension was passed through a 100μm strainer, pelleted at 300 g for 6 minutes, suspended in 2mL RBC (Red Blood Cell) Lysis Buffer (BioLegend) and incubated for 2 min. At this point, 8mL HBSS was added and centrifuged again. The pellet was suspended in HBSS, filtered through a 30um strainer and pelleted and suspended in MACS separation buffer (Miltenyi Biotec) with 2% FBS for FACs and in HBSS/0.01% BSA for 10x single cell sequencing. Cell separation and viability were confirmed under the microscope and through Vi-CELL Cell Counter after staining with Trypan blue.

Wild-type C57Bl/6 mice underwent ischemia/reperfusion injury (IRI) at 7 weeks for 30 min as previously described [40 (link)]. Mice were sacrificed at 3 days (n = 3) and 7 days (n = 3) after IRI. Healthy mouse kidney (n = 1) was used as control. At the time of sacrifice, kidneys were minced into 1 mm pieces and incubated at 37 °C for 10 min in a buffer containing 250 U/mL Liberase (Roche, Mannheim, Germany) and 40 U/mL DNase I (Sigma-Aldrich, Taufkirchen, Germany). The obtained solution was then transferred to a Miltenyi C-tube, and the gentleMACS D1 program was run (Miltenyi Biotec, Bergisch Gladbach, Germany). The steps were repeated twice. The reaction was stopped by adding 10% FBS, and the dissociated cells were passed through a 40 µm cell strainer and incubated with RBC lysis buffer. Dead cells were removed employing the dead cell removal kit (Miltenyi Biotec, Bergisch Gladbach, Germany) and run on a 10x Chromium Single Cell instrument (10× Genomics). This method generated single cell suspension with greater than 95% viability.

Murine tissues (fetal membranes and placenta) collected after experiment were digested with 1mL of Accutase (Innovative cell technologies) for 30 minutes at 37°C with gentle shaking. The tissues were transferred into a C-tube (Miltenyi Biotec) H-cord-01 setting. The tissue was washed with 20mL of 1x PBS, filtered through a 40-µm, and centrifuged at 1250g for 10 minutes at 4°C. Cell pellets were incubated in 5mL of red blood cells (RBC) lysis buffer (biolegend) for 10 minutes at room temperature. Cells were centrifuges at 1250g for 10 minutes at 22°C. The cell pellets were resuspended in the Maxpar staining buffer for further analysis.