Facsaria fusion cell sorter

Whole blood was collected in ethylene diamine tetraacetic acid (EDTA) tubes, and peripheral blood mononuclear cell (PBMC) preparations have been performed as described previously (23 (link)). PBMCs were cryopreserved in freezing medium (heat-inactivated fetal calf serum (FCS), PAN-Biotech; 10% v/v dimethyl sulfoxide, Sigma) until use. Upon cell thawing, PBMCs were rested overnight in complete RPMI medium (at 37°C, 5% CO₂) for recovery and collected in the morning for further processing. After washing once in fluorescence-activated cell sorting (FACS) buffer [phosphate buffered saline (PBS), 5% v/v FCS], the cells were stained with antibodies in the presence of Octagam 10% (Octapharma). PBMCs were stained with CD4-BV421 (clone RPA-T4, BD Biosciences), CD25-BV510 (clone 2A3, BD Biosciences-OptiBuild), and CD127-PE (clone A019D5, BioLegend) antibodies and Fixable Viability Dye eFluor780 (Thermo Fisher). Regulatory T cells (Tregs) (live CD4⁺ CD25^hi CD127^lo) were sorted under aseptic condition in BD FACSAria Fusion cell sorter (Becton-Dickinson). Cell sorting typically yielded high enrichment (>90%) of Tregs. Sorted Tregs were stimulated with anti-CD3/CD28 beads (Dynabeads, Thermo Fisher) for 16 h. Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) expression was assessed in Cyto-Fast Fix/Perm (BioLegend)-permeabilized cells using a CTLA-4-APC antibody (BioLegend).

Flow cytometry and cell sorting was performed using a BD FACSAria Fusion Cell Sorter (Becton, Dickinson and Company) with FACSDIVA software v8.0.1. Sorting of single cells into 96-well plates was performed using the automated cell deposition unit. Verification of the single-cell sorting mode was established by sorting single fluorescent beads (Alignflow Flow Cytometry Alignment Beads; Thermo Fisher Scientific) into a flat-bottomed 96-well tissue culture plate. A fluorescence microscope was used to visualize the beads to verify that no wells contained more than one fluorescent bead and that they were centrally positioned in the wells. Further flow cytometry analysis was performed using FlowJo software v10.7 (Becton, Dickinson and Company).
Cryopreserved peripheral blood mononuclear cells were thawed and processed for flow cytometry analysis and cell sorting as previously described^{33 (link)}. Staining panel details for granulocyte, T cell and HSPC analysis and sorting are shown in Supplementary Table 8, and the gating strategy is provided in Supplementary Fig. 1.

Following 8–14 weeks post orthotopic transplants, or ~8 weeks post i.c. or i.v. injections, primary tumors and peripheral tissues including lungs, lymph nodes, bone marrow, peripheral blood, and brains were harvested, dissociated to single cells and stained with fluorescently conjugated antibodies for CD298 (Biolegend, Cat. no. 341704) and MHC-I (eBioscience, Cat. no. 17-5957-82), and flow cytometry was used to analyze and quantify disseminated CD298⁺MHC-I⁻ PDX cells using the BD FACSAria Fusion cell sorter (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) as described previously^{26 (link)}. Primary tumor volumes were calculated using caliper measurements with the equation: volume of an ellipsoid = 1/2(length × width²). Whole-mount images of primary tumors and lungs were taken with a Leica MZ10 F modular stereomicroscope (Leica Microsystems, Buffalo Grove, IL, USA).

Full‐length wild‐type ECE1c cDNA with in‐frame 5′‐Flag and Myc tags was inserted into the multicloning region of plasmid pLVX‐IRES‐mCherry (Clontech, Mountain View, CA, USA). Site‐directed mutagenesis was performed using the GENEART kit (Invitrogen) according to manufacturer’s instructions. Lenti‐X 293T cells were transfected with 8 μg psPax2 (encoding Gag‐Pol protein), 8 μg pLVX‐IRES‐mCherry (encoding ECE1c^WT, ECE1c^K6R or empty), and 4 μg pCMV‐VSVg (encoding VSV G‐glycoprotein) and then suspended in 500 μL 0.25 m CaCl₂. At 48 h post‐transfection, supernatants containing pseudotyped particles were harvested and passed through a cellulose acetate filter with a pore size of 0.45 μm. Viral particles were purified and concentrated by ultracentrifugation at 150 000 g for 75 min in SureSpin 630 rotor (Thermo Fisher, Vilnius, Lithuania) through a 25% sucrose cushion (TNE‐Sucrose 25%). Finally, cells were cultured at 5 × 10⁴ cells/well in 12‐well plates along with the recombinant lentiviruses at a MOI of 5 under normal growth conditions. Expression of mCherry was examined 72 h post‐transduction under a Nikon Eclipse TS100 Inverted Microscope (Nikon, Tokyo, Japan) equipped with epifluorescence. Cells were expanded for 1 week, and the brightest (mCherry⁺) cells were sorted on a FACSAria Fusion cell sorter (Becton‐Dickinson, San Jose, CA, USA).

Preparation of FACS tissue was carried out as previously described [62 ]. Cortex from perfused brains was mechanically homogenized and enzymatically digested before straining and separating through a Percoll gradient. Antibodies for CD3 (APC hamster anti-mouse CD3; 1:200; BD Bioscience #553,066), CD11b (PE rat anti-CD11b; 1:200; BD Bioscience #557,397), and CD45 (FITC rat anti-mouse CD45; 1:100; BD Bioscience #553,079) were added to cell suspensions and whole blood samples before the samples were washed and centrifuged, and the pellet resuspended in flow cytometry buffer. Samples were sorted on a FACS Aria™ Fusion cell sorter (Becton Dickenson), lymphocytes were identified as CD3 + , and macrophages were identified as being CD11b + CD45hi.

Cells were grown in 6-well plates, washed with PBS, and trypsinized in 100-μl 0.05% Trypsin-EDTA. The cells were collected in 1-ml PBS supplemented with 1% FBS and passed through the cell strainer cap of the FACS test tubes (Falcon). The cells were kept on ice until measurement on FACSAria FUSION cell sorter (Becton Dickinson). Data were analyzed, and figures were created using FlowJo (FlowJo, LLC).