Facs diva software

To analyze early changes in the TIME, mice were injected with 5 × 10⁵ tumor cells via the intrasplenic/portal route, and the livers removed 3, 7, or 9 days later (as indicated). Liver homogenates were prepared in cold PBS and filtered through a stainless steel mesh using a plunger. The filtrates were centrifuged at 60 G to separate the hepatocytes, the supernatants containing the nonparenchymal cell fraction centrifuged at 480 g and the pellets resuspended in 10 ml of a 37.5% Percoll solution in HBSS containing 100 Uml⁻¹ heparin and centrifuged at 850 g for 30 min to obtain the immune cell-rich fraction. Prior to FC, red blood cells were removed using the ACK (ammonium–chloride–potassium) solution and 1 × 10⁶ cells were immunostained with the indicated antibodies. Data acquisition was with a BD Canto flow cytometer and FACS Diva software and the data analyzed using the FlowJo software. For flow cytometric experiments on hepatic leukocytes, single cells were gated based on size (forward scatter), granularity (side scatter) and viability using an eFluor™ 780 fixable dye (eBioscience™, ThermoFisher).

Small intestinal crypts and villi were processed as described above, with an additional step of mild pipetting using DPBS/5% FBS solution following EDTA chelation to increase crypt cell yield. Single-cell suspensions were then obtained from enriched crypts and villi by incubation in culture media containing ROCK inhibitor for 45 min at 37 °C, followed by mild mechanical dissociation using a syringe with a 21G needle^{58 (link)}. The suspension was then filtered through a 40 µm mesh and dissociation and cell count determined using a hemocytometer. Stainings were performed with fluorophore-conjugated antibodies in 2% FBS/PBS solution for 25 min on ice in the dark (see Supplementary Table 2 for used antibodies). Live/dead staining has been achieved by addition of DAPI right before data acquisition on a BD Fortessa flow cytometer. Flow cytometry assisted cell sorting (FACS) has been performed using FACS Aria II. FACS Diva Software and FlowJo V10 has been employed to process and analyze data.

Reovirus virions (3 × 10¹² particles) were incubated with 20 μM Alexa Fluor 647 NHS ester (Invitrogen, #A20006) and 50 mM NaHCO₃ in a 500 μl total volume rotating at room temperature (RT) for 1.5 h¹⁹ . Unconjugated fluorophore was removed by dialysis overnight in phosphate-buffered saline (PBS) buffer at 4 °C. For reovirus binding assays, cells were dissociated with Cellstripper, incubated with labeled reoviruses (2 × 10⁵ virions/cell) at 4 °C for 1 h, and washed three times with ice-cold 2% FBS DMEM. For PirB-specific antibody blockade assay, CHO cells were incubated with PirA/B-specific monoclonal antibody (mAb) 6C1 (BioLegend, #144101) or isotype IgG (BioLegend, #400402) at 4 °C for 1 h and washed twice with PBS prior to reovirus binding. For cell sorting during the CRISPRa screen, unfixed living MEFs were analyzed using a FACSAria II cell sorter (BD Biosciences) operated by FACSDiva™ Software (BD Biosciences, v6.1.3). For other reovirus binding assays, cells were fixed with 1% paraformaldehyde (PFA, Electron Microscopy Sciences) at 4 °C overnight. Reovirus-binding on cells were measured using an LSR II flow cytometer (BD Biosciences) operated by FACSDiva™ Software (v6.1.3) and analyzed by FlowJo software (v10.8.1).

K562 cells that do not express the Major Histocompatibility Complex (MHC) class I molecules on the cell surface (missing self) were used as classical targets of NK cells to evaluate their cytotoxic activity as previously described (4 (link)). Briefly, K562 cells were stained with PKH Red Fluorescence Cell Linker kit (Sigma-Aldrich Merck, Darmstadt, Germany) and then cocultured with PBMCs (1:2). After 1 h, they were collected and stained with Annexin V conjugated with fluorescein isothiocyanate (FITC) (Thermo Fisher, Waltham, MA, USA) to quantify early apoptosis by measuring the expression of phosphatidylserine on the cell surface by flow cytometry. BD LSRFortessa X-20 flow cytometer and FACS Diva software were used for data acquisition, and FlowJo_V10 software was used for data analysis.

Flow cytometry used to assess cell surface expression of PSGL-1 was performed using a BD LSRFortessa (San Jose, CA, USA) instrument with FACS Diva software (San Jose, CA, USA), and all data were analyzed using FlowJo software version 10.7.1. (San Jose, CA, USA). T cell lines or activated PBMC were stained before infection with an isotype control antibody or a mouse anti-human monoclonal antibody against PSGL-1 (same clone as above) for 45 min. After primary antibodies were removed by washing, staining with an R-phycoerythrin (PE) conjugated F(ab’) 2-goat anti-mouse IgG secondary antibody (Invitrogen, Carlsbad, CA, USA; Cat#A10543) was performed for 20 min. HEK293T cells were stained following the same protocol 48 h after transfection. All staining was performed with 2 µg/mL of antibody in 4 °C in the dark.