Dead cell removal kit

Tissues obtained from hysterectomies included endometrium (EM), endocervix (CX) and ectocervix (ECX) and were transferred to the laboratory immediately after surgery and processed as previously described (3 (link), 6 (link), 15 (link), 17 (link), 18 (link)). Vaginal tissues were not available. Tissues were rinsed with HBSS and minced under sterile conditions into 1–2 mm fragments and digested using an enzyme mixture containing 0.05% collagenase type IV (Sigma-Aldrich, St. Louis, MO) and 0.01% DNAse (Worthington Biochemical, Lakewood, NJ) for 1h at 37°C. Type IV collagenase was selected based on preliminary studies to ensure non-cleavage of surface markers (3 (link), 15 (link)). After digestion, cells were dispersed through a 250 μm mesh screen (Small Parts, Miami Lakes, FL) and filtered through a 20 μm mesh screen (Small Parts) to separate epithelial cells from stromal cells. Stromal cells were then washed and counted and dead cells removed using the Dead cell removal kit (Miltenyi Biotec, Auburn, CA) according to manufacturer instructions to obtain a mixed cell suspension for flow cytometric analysis, degranulation assays and further cell purification.

The tumors were resected at indicated time points, and extracted tumors were maintained in ice-cold PBS supplemented with 0.5% BSA until further use. The single-cell suspension was generated using Tumor Dissociation Kit, mouse (Miltenyi Biotec) as per the manufacturer’s protocol. Live cells from single cell suspension were enriched using Dead Cell Removal Kit (Miltenyi Biotec) according to the manufacturer’s protocol. The cells were then stained with Fixable Viability Stain (FVS780) at room temperature. The cells were labeled at 4°C with respective antibodies. TAM were isolated using BD FACSAria III cell sorter (BD Biosciences).

CD8+ T cells were purified from the mixed cell suspension using a negative magnetic bead selection kit (Miltenyi Biotec, Auburn, CA) as previously described by us (17 (link), 23 (link)). Dead cells were removed using the Dead cell removal kit (Miltenyi Biotec). To remove stromal fibroblasts from the mixed cell suspension anti-fibroblasts microbeads were also added. Purity of the CD8+ T cell population was higher than 90% following two rounds of negative selection (17 (link), 23 (link)).
To separate CD103-CD8+ T cells from CD103+CD8+ T cells, the purified CD8+ T cell suspension was incubated with CD103-PE antibody (Miltenyi Biotec) for 10 minutes after which anti-PE ultra-pure magnetic beads (Miltenyi Biotec) were added. CD103- were separated by negative selection and CD103+ by positive selection resulting in a purity range between 90-95% for CD103- and CD103+ CD8+ T cells respectively (17 (link), 23 (link)).

All the included patients were pathologically diagnosed with lung adenocarcinoma. About 1cm³ of tumor tissue and tumor adjacent tissue during curative surgery were collected and dissociated into single-cell suspensions by Tumor Dissociation Kit (130-096-730, Miltenyi). Cell suspensions were added with ACK lysis buffer (A10492-01, Gibco) to lyse the remnant RBC, and treated with Dead Cell Removal Kit (130-090-101, Miltenyi), then filtered through the 40-μm plastic mesh (Falcon). Cell suspensions were stained with DAPI for viability, and living cells were sorted with a BD FACSAria III instrument.

DCs isolated from four C57BL/6J mice were used for each experimental group. DCs were cocultured for 6 h with dying/dead glioma GL261 cells in a ratio of 1:5. There were three experimental groups: DCs alone (negative control) and DCs cocultured with glioma GL261 cells pulsed with PS-PDT (PS at a dose of 1.4 µM, 24 h) or with MTX (positive control, 2 µM, 24 h). For the cocultures of DCs with treated GL261 cells, additional technical replicates were included. After 6 h of coculture, the cells were harvested and washed once with DPBS. Enrichment of DCs from the coculture proceeded as follows. First, dead cells were removed with a Dead Cell Removal Kit (MACS Miltenyi Biotec). Then, the final eluate was loaded onto MS columns (MACS Miltenyi Biotec) and treated with CD11c MicroBeads UltraPure (MACS Miltenyi Biotec) to enrich for CD11c⁺ DCs. The purity of CD11c⁺ DCs was analyzed by flow cytometry on a BD FACS Canto II flow cytometer after each step. Enriched CD11c⁺ cells were snap frozen in liquid nitrogen, and total RNA was isolated using RNeasy Mini Kit (QIAGEN). The quantity and integrity of the RNA were determined with a NanoDrop 8000 spectrophotometer (Thermo Fisher) and a Fragment Analyzer 5200 system (Agilent), respectively.

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.