Ebioscience ic fixation buffer

Expression of oxMIF at the cell surface of human prostate adenocarcinoma cell line PC-3 (RRID:CVCL_0035, ECACC, Sigma catalog no. 90112714) and colon carcinoma cell line HCT116 (RRID:CVCL_0291, ECACC, Sigma catalog no. 91091005) was assessed by flow cytometry. Cells were dislodged from flasks by treatment with CellStripper nonenzymatic cell dissociation buffer (VWR, catalog no. 25-056-CI) and were stained with anti-oxMIF mAbs, or isotype IgG (palivizumab, Abbvie, catalog no. PZN-10974950) at 75 nmol/L in PBS supplemented with 5% BSA for 45 minutes at 4°C. After washing with PBS+5% BSA, cells were stained with a polyclonal goat anti-human IgG (H+L) Alexa Fluor488-conjugated antibody (Thermo Fisher Scientific, catalog no. A11013, RRID:AB_2534080) diluted 1:200 in PBS+5%BSA for 40 minutes at 4°C for the detection of oxMIF-bound mAbs. Cells were next washed with PBS and stained with eBioscience Fixable Viability Dye eFluor 780 (Thermo Fisher Scientific, # 65-0865-14) diluted in PBS (1:2,000) for 20 minutes at 4°C. After washing with PBS supplemented with 5% BSA, cells were fixed with eBioscience IC Fixation Buffer (Thermo Fisher Scientific, catalog no. 00-8222-49) for 10 minutes at 4°C. After washing, cells were measured on a CytoFlex-S flow cytometer (Beckman Coulter, RRID:SCR_019627, and data were analyzed by using FlowJo software (BD, RRID:SCR_008520).

BMDMs were seeded on 13 × 1.5 mm round coverslips (VWR International) at 1 × 10⁵ cells in a 24‐well plate and rested overnight. Cells were treated as described in figure legends. After treatments, BMDMs were washed in PBS and fixed in eBioscience™ IC fixation buffer (Thermo Fisher Scientific) for 10 min at RT. BMDMs were permeabilised in 0.1% Triton X‐100 in PBS for 4 min, washed and then blocked in 0.4% gelatin from cold water fish skin (Sigma‐Aldrich) in PBS for 10 min. Following incubation in 0.1% sodium borohydride pH 8.0 (Sigma‐Aldrich) for 10 min, cells were stained with primary antibody solution (1:200) for 1 h, followed by three washes. BMDMs were incubated in secondary antibody solution (1:1,000) for 30 min. Nuclei were stained by incubation with 4′,6‐diamidino‐2‐phenylindole (DAPI) (1:1,000) for 15 min. Washed coverslips were mounted on Corning microscope slides (Merck) using Fluoromount mounting solution (Sigma‐Aldrich). Slides were visualised under a Zeiss Inverted LSM 880 Axio Observer confocal laser scanning microscope (ZEISS) using a ZEISS 40× Plan‐Apochromat (numerical aperture = 1.3) DIC M27 oil immersion objective. Where indicated, a ZEISS 63x Plan‐Apochromat (numerical aperture = 1.4) DIC UV‐VIS‐IR M27 oil immersion objective was used.

The SIINFEKL peptide (H-2K^b-restricted) was synthesized and HPLC purified (95% purity) at the HZI peptide-synthesis platform. For intracellular staining, single cell suspensions were incubated with 1 μg/ml SIINFEKL peptides in complete RPMI medium for 1 h at 37°C followed by brefeldin A (Golgiplug; BD Pharmingen) addition and further 5 h incubation. For intracellular staining, surface-labelled cell suspensions were fixed using eBioscience Foxp3/Transcription Factor Staining Buffer Set or eBioscience IC Fixation Buffer (both from Thermo Fisher). Cell suspensions were stained with following antibodies at 4°C for 30 min: anti-GzmB (clone QA16A02), anti-IFN-γ (clone XMG1.2) and anti-TNF-α (clone MP6-XT22). After washing with FACS buffer, the cells were analyzed using flow cytometer.

Cell suspensions were stained for viability using LIVE/DEAD Aqua dye (Life Technologies). Cells were washed and resuspended in 50 μl of blocking buffer containing TruStain FcX PLUS (anti-mouse CD16/32, clone S17011E) antibody diluted in PBS and incubated in the dark for 15 min on ice. Conjugated anti-mouse antibodies CD45 (30-F11), CD9 (eBioKMC8), CD29 (HMβ1-1), CD31 (390), and Integrin α 7 (334908), Sca-1 (D7) were used to stain cell surface markers for 30 min on ice. Finally, cells were fixed with 50 μl of eBioscience IC Fixation Buffer (Thermo Fisher Scientific) for 5 min and acquired using a 5-laser LSR II flow cytometer (BD Biosciences) with BD FACSDiva software. Data were analyzed with FlowJo v10.6.2 (Becton, Dickinson and Company).

Mice were anesthetized by inhalation with isoflurane, and blood from the hearts of mice was placed in heparin-coated tubes and treated with RBC lysis buffer. The following monoclonal antibodies were used for flow cytometric analysis at a dilution of 1/400: anti-CD45, anti-CD11b, anti-Ly6G, anti-Ly6C, anti-Ly6G/Ly6C (Gr-1) antibodies. For intracellular staining, after staining with antibodies against cell surface proteins, cells were washed three times with MACS buffer, fixed with eBioscience™ IC Fixation Buffer (Thermo Fisher Scientific), permeabilized with eBioscience™ Permeabilization Buffer, and then stained with an antibody against an intracellular marker. Cells not stained with FVD780 were categorized as live cells. Data were acquired with LSRFortessa (BD Biosciences, San Jose, CA, USA) and analyzed with FlowJo. The gating strategies of neutrophils (CD45⁺CD11b⁺Ly-6G^intra+) are shown in Supplementary Figure S4.