Intracellular staining permeabilization wash buffer

For flow cytometry staining, cells were washed and stained in PBA buffer (0.5% BSA, 0.02% sodium azide in PBS) for 30 min on ice. For intracellular cytokine staining, cells were fixed and permeabilised with Intracellular Staining Permeabilization Wash Buffer (Biolegend, San Diego, CA, USA). Antibodies used were: PE-Cy7 anti-CD86 (Cat. No. 560582, 1:100), PE-Cy7 anti-IFNγ (Cat. No. 557649, 1:200), both from BD Pharmigen, San Diego, CA, USA), APC-R700 anti-CD8 (Cat. No. 564983, 1:500), V500 anti-I-A/I-E (Cat. No. 562366, 1:1000, all from BD Horizon, Franklin Lakes, NJ, USA), PE-Dazzle 594 anti-TNFα (Cat. No. 506345, 1:100), PerCP-Cy5.5 anti-CD8a (Cat. No. 100733, 1:500), PerCP-Cy5.5 anti-CD172a (Cat. No. 144009, 1:100), BV711 ant-CD25 (Cat. No. 10204, 1:200), BV605 anti-Ly6C (Cat. No. 128035, 1:1000), BV786 anti-XCR1 (Cat. No. 148225, 1:100), BV711 anti-CD64 (Cat. No. 139311, 1:100), BV650 anti-CD19 (Cat. No. 115541, 1:100), Alexa Fluor 700 anti-CD45.1 (Cat. No. 110723, 1:100, all from Biolegend, San Diego, CA, USA), eF450 anti-CD45.1 (Cat. No. 48-0453-82, 1:100), PE anti-CD103 (Cat. No. 12-1031-82, 1:200), Alexa Fluor 700 anti-CD4 (Cat. No. 56-0041-82, 1:200), Fixable Viability Dye eFluor780 (Cat. No. 65-0865-14, 1:1000 all from eBioscience, San Diego, CA, USA). APC or PE labeled H-2K^b-SIINFEKL tetramers were made in-house and used at dilution 1:200.

Expanded γδ T cells from colon tumours were analysed by flow cytometry for the expression of TCR Vδ chains, NKG2 receptors, NCRs, KIRs, tissue-residency/activation markers, cytotoxic molecules, immune checkpoint molecules, cytokine receptors and Fc receptors. In brief, cells were incubated with human Fc receptor block (BioLegend) and stained with cell surface antibodies (Supplementary Table 7) for 45 min at 4 °C, followed by three wash steps in FACS buffer (PBS/1% FCS). Granzyme B and perforin were detected intracellularly using fixation buffer and intracellular staining permeabilization wash buffer (BioLegend). Compensation was performed using CompBeads (BD Biosciences) and ArC reactive beads (Life Technologies). Cells were acquired on the FACS LSR Fortessa 4L (BD Biosciences) system running FACSDiva software (v.9.0; BD Biosciences). Data were analysed using FlowJo (v.10.6.1; Tree Star).

Cultured hippocampal neurons were fixed on coverslips with a Fixation Buffer (#420801, BioLegend, San Diego, CA, USA), permeabilized with an Intracellular Staining Permeabilization Wash Buffer (#421002, BioLegend), then blocked with 0.2% Triton X-100, 3% fetal bovine serum (#26140087, Gibco) and 1% DMSO. Immunofluorescence staining was carried out by incubation for 48 h at 4 °C using a rabbit polyclonal antiserum against VEGFR2 (1:250; #ab2349, Abcam, Cambridge, MA, USA) and a mouse monoclonal antiserum against β-Tubulin III (1:1000; #T8578, Sigma-Aldrich). This was followed by incubation with a secondary goat anti-rabbit IgG conjugated with Alexa Fluor 488 (1:500; #A11034, Invitrogen, Eugene, OR, USA) and a donkey anti-mouse IgG conjugated with Alexa Fluor 568 (1:500; #A10037, Invitrogen) for 1 h at room temperature. Nuclei were stained in a commercial mounting medium that contains 0.0002% DAPI (#ab104139, Abcam). Slides were viewed under an Olympus FV10i confocal microscope (Olympus, Tokyo, Japan). FV10i-SW software, 60× objective and 405, 473 and 559 nm laser lines were used for image acquisition; immunoreactivity for β-Tubulin III exhibited red fluorescence, and VEGFR2 exhibited green fluorescence.

Splenocytes were incubated with TruStain FcX™ PLUS anti-mouse CD16/CD32 (Biolegend; clone: S17011E) for 10 min at 4°C, and then stained with mAbs specific for various cell surface markers. To evaluate the production of IFN-γ and Granzyme B, splenocytes were diluted to 4 × 10⁶ cells/mL and cultured (500 μL/well) in a 24-well plate containing Cell Activation Cocktail (with Brefeldin A; Biolegend) for 6 h. Cells were then harvested and washed twice in cell staining buffer prior to surface marker staining as described above. Cells were then fixed and permeabilized using Intracellular Fixation Buffer & Intracellular Staining Permeabilization Wash Buffer (Biolegend). Intracellular staining was then performed using mAbs specific for IFN-γ and granzyme B, respectively. For Ki-67 staining, freshly isolated splenocytes were stained following the above-mentioned surface and intracellular staining procedures, with no in vitro stimulation. Samples were resuspended in cell staining buffer, tested with BD FACSCelesta flow cytometer, and analyzed using FlowJo software (BD, USA).

Anti-mouse Ly6G (300 μg; clone IA8; BP0075-1; BioXCell) or its isotype control rat IgG2a (clone 2A3; BP0089; BioXCell) in 1× PBS was administered via the i.p. route to LPS/saline mice. Antibody injections began the same day as LPS/saline injections and continued every 48 h. CFU numbers were assessed at 7 days of infection. Single-cell suspensions were isolated at day 0 (uninfected) and analyzed by flow cytometry to assess depletion efficiency. Intracellular Ly6G was used in place of surface Ly6G to account for potential surface antigen masking by the depletion antibody (70 (link)). After 2% PFA fixation, intracellular Ly6G was stained in the intracellular staining permeabilization wash buffer (BioLegend) using the manufacturer’s protocol. Ly6G antibodies (Ly6G, PE; clone 1A8; BD Pharmingen) used for intracellular staining were diluted half of what was typically used for surface staining.

The cell cycle was analyzed in control fibroblasts using FxCycle^TM Far Red Stain (Thermo Fisher Scientific) 48 h post transfection. After trypsinization and centrifugation, cells were washed in PBS, centrifuged, and fixed with a fixation buffer (Biolegend, San Diego, USA) for 20 min at room temperature. Cells were washed with Intracellular Staining Permeabilization Wash Buffer (1/10, Biolegend), centrifuged and permeabilized using the same solution for 20 min at RT. After the last centrifugation, cells were resuspended in 1 mL PBS containing 1 μL FX Cycle Far Red and 5 μL Ribonuclease A (Sigma) and incubated for 30 min at RT. Cell cycle distribution was analyzed on an Attune cytometer (Thermo Fisher). Results were analyzed using FlowJo software (FlowJo software, Oregon, USA).