Fluorescein isothiocyanate (fitc)

The isolated colonic lamina propria and intestinal epithelial cells were stained with protein markers used to delineate macrophages and intestinal epithelial cells, respectively. Cells were stained with cell surface markers for 30 min in flow buffer, followed by fixing with 4% paraformaldehyde and permeabilization for intracellular staining. Macrophages producing Tnfα were marked as CD45⁺F480⁺CD11b⁺Tnfα⁺, while resident macrophages expressing Cx3Cr1 producing Tnfα were marked as CD45⁺F4/80⁺CD11b⁺Cx3cr1⁺Tnfα⁺. Epithelial cells were marked as EpCam⁺, while EpCam⁺CD24^lo was used to characterize cycling epithelial cells. Antibody dilutions were as follows: CD45 1:100 (Alexa Fluor 700; BioLegend, San Diego, CA, USA), F4/80 1:50 (APC; Miltenyi Biotec, Bergisch Gladbach, Germany), CD11b 1:100 (Fitc; BioLegend, San Diego, CA, USA), Cx3Cr1 1:100 (PE, BioLegend, San Diego, CA, USA) Tnfα 1:50 (Pe-Cy7; BD Biosciences, San Jose, CA, USA), EpCam 1:100 (Fitc; BioLegend, San Diego, CA, USA), CD24 1:100 (PE; BioLegend, San Diego, CA, USA).

All antibodies used in this study were purchased from BD Biosciences unless stated otherwise. The clone numbers are indicated in parentheses. PBMC were harvested for flow cytometry after 24 and 48 h post-establishment of the co-culture. Cells were first washed with PBS and incubated at room temperature for 15 min with Fixable Live/Dead Blue Viable Dye (Thermo Scientific). After incubation, the cells were washed in PBS buffer containing 0.5% BSA and 2 mM EDTA. Subsequently, the cells were incubated with the following antibodies: Vδ1 TCR (TS-1, Thermo Scientific) FITC, Vδ2 TCR (B6, Biolegend) PerCP, CD3 (UCHT1) V500, CD8 (SK1) APC-Cy7, CD14 (MϕP9) PE-CF594, CD56 (B159) PE-Cy7, CD69 (FN50, Biolegend) BV421, CD83 (HB15e) APC, CD161 (HP-3g10, Biolegend) BV605, Vα7.2 (3C10, Biolegend) PE, and CD38 (HB7) BUV395 for 15 min at 4°C. Assays on NK cells were performed additionally with the following antibodies: CD3 (UCHT1) V500, CD16 (3G8, Biolegend) FITC, CD56 (B159) PE-Cy7, CD38 (HB7) BUV395, CD69 (FN50 Biolegend) PE. Flow cytometry analyses were performed using a BD LSRII flow cytometer (BD). Subsequent gating analyses were performed using FlowJo software version 9.9.6 or 10.5.2 (Tree Star Inc.).

For phenotyping human circulating monocytes, peripheral blood mononuclear cells (PBMCs), which contain lymphocytes and monocytes, but no neutrophils, were obtained from the anticoagulated peripheral blood of TBI patients or patients who underwent cranioplasty by centrifugation with Ficoll-Paque Plus (Sigma-Aldrich, MO, USA). PBMCs were stained with anti-human CD16 (Clone: 3G8)-FITC (BioLegend, CA, USA, Cat: 302006), CD14 (Clone: MΦP9)-PE (Becton Dickinson, NJ, USA, Cat: 347497), and CXCR2 (Clone: 5E8/CXCR2)-APC (BioLegend, Cat: 320710) fluorescent monoclonal antibodies (mAb). For phenotyping monocytes in the CSF, 10 mL of anticoagulated CSF was obtained from TBI or non-TBI patients. Nucleated cells were collected by centrifugation at 150 g for 10 min, and then stained with anti-human CD11b (Clone: ICRF44)-FITC (BioLegend, Cat: 301330), CD14 (Clone: MΦP9)-PE (Becton Dickinson, Cat: 347497), and CXCR2 (Clone: 5E8/CXCR2)-APC (BioLegend, Cat: 320710) fluorescent mAb. To analyze surface calreticulin (CRT) expression, SH-SY5Y cells were stained with anti-human CRT (Clone: 1G6A7)-APC (Novus Biologicals, CO, USA, Cat: NBP1-47518APC). The isotype controls were analyzed in parallel. The samples were acquired on a FACSCalibur flow cytometer (BD Biosciences, NJ, USA) and analyzed using FlowJo software (FlowJo, OR, USA).