Mouse igg1

CM were collected at day 14 of differentiation for flow cytometry analysis. Briefly, cells were washed with PBS and treated with TrypLE (ThermoFisher) for 5–10 min at 37 °C and collected in RPMI. Cells were spun down and resuspended in 200 μL 4% paraformaldehyde for 10 min at RT. Cells were spun down and resuspended in 500 μL PBS + 5% FBS and split into two tubes for staining. Tubes spun 2,000 rpm × 5 min and were resuspended in 50 μL PBS + 5% FBS + 0.75% saponin with either 0.5 μL mouse IgG1 (eBioscience 14-4714) or 0.5 μL mouse cTnT (ThermoFisher MS-295) and incubated for 30 min at RT. After incubation 150 μL PBS + 5% FBS + 0.75% saponin were added and samples were spun down and washed with 200 μL PBS + 5% FBS + 0.75% saponin. Both sets were resuspended after washing in 50 μL PBS + 5% FBS + 0.75% saponin with 0.25 μL goat anti-mouse PE secondary (Jackson 115-116-072) and incubated for 30 min RT in the dark. Following secondary 150 μL PBS + 5% FBS were added and samples were spun down and washed with 200 μL PBS + 5% FBS. Samples were then stored in 400 μL PBS + 5% FBS + 100 μL 4% paraformaldehyde at 4 °C until analysis. Samples were run on a BD FACSCanto II and data were acquired with the BD FACSDIVA software (both from BD Biosciences). Analysis was performed with FloJo version 10 software. Only samples >75% cTnT positive were used for downstream applications.

HUVEC, HMEC-1 or HC-6014 were separated from their co-culture with Caki-1 cells. For separation by flow cytometry Caki-1 cells were marked with the CellTracker CM-Dil dye (Mo Bi Tec, Göttingen, Germany) prior to co-culture (detectable by FACScalibur, BD Biosciences, Heidelberg; FL2-H channel histogram analysis; 1 × 10⁴ cells/scan). Cells were detached, washed in FACS buffer (PBS, 0.5% BSA) and incubated for 60 min at 4°C with monoclonal antibodies (mAbs) directed against the following endothelial adhesion receptors: ICAM-1 (CD54, HA58), VCAM-1 (CD106, 51-10C9), E-selectin (CD62E, 68-5H11), P-selectin (CD62P, AK-4, all: APC labelled, anti-human, mouse IgG1 K, 20 μl, BD Pharmingen, Heidelberg, Germany), CD44 standard (CD44std, SFF-2, mouse IgG1 K, 1:20) and CD44 variants V3 (VFF-327v3, 1:10), V4 (VFF-11, 1:20), V5 (VFF-8) and V7 (VFFF-9, 1:20, all: mouse IgG1, 20 μl, eBioscience, Frankfurt, Germany). All CD44 antibodies were labelled with APC (Lightning-Link Allophycocyanin – XL Conjugation Kit, Innova Biosciences, Cambridge, UK). APC mouse IgG1, K (MOPC-21, 20 μl, BD Pharmingen, Heidelberg, Germany) served as isotype control. Receptor expression of HUVEC was measured by flow cytometry using FACScalibur (BD Biosciences, Heidelberg; FL4-H channel histogram analysis; 1 × 10⁴ cells/scan). Data were expressed as mean relative fluorescence intensity (MFI).

The expression of Mac-1 and LFA-1 was assessed by flow cytometry as described previously [11 (link)]. After red blood cell lysis, 10⁵ cells were incubated in the dark with 2 μg FITC-conjugated anti-LFA-1 or anti-Mac-1 (eBioscience, San Diego, USA) or 2 μg FITC-conjugated isotype control antibodies (Mouse IgG1, eBioscience, San Diego, USA). Mac-1 and LFA-1 expression was assessed on 10.000 cells per mouse within the neutrophil cluster defined by forward-sideward scatter analysis using LSRII with DIVA software package (Becton Dickinson, San Jose, USA). Expression of Mac-1 and LFA-1 was compared to their respective isotype controls.
For differentiation of cells before and after isolation procedures flow cytometry was performed on 10⁵ unstained cells using standard neutrophil, monocyte and lymphocyte clusters defined by forward-sideward scatter analysis.

Thirty-six male clean-grade Wistar rats (6 weeks old, 100–120 g) of inbred lines were obtained from Vital River Experimental Animal Technology Co., Ltd. (Beijing, China) and randomly divided into four groups as follows: the normal control group, IL-6-blocked control group, IR control group, and IR IL-6-blocked group. IR was induced by feeding the rats a sterilized high-fat diet rich in unsaturated fatty acids (fat providing 59% of the calories) for 28 days, and then insulin sensitivity was quantified by the euglycemic hyperinsulinemic clamp technique. To block IL-6, 8 mg of anti-mouse IL-6 receptor antibody (MR16-1; Chugai Pharmaceutical Co., Ltd., Japan) was intraperitoneally injected into each rat on days 0 and 14 after IR model establishment. The same dose of mouse IgG1 (eBioscience, Inc., San Diego, CA, USA) was administered to the control group. After 4 weeks, the rats were killed, and samples of adipose tissue and blood were obtained for testing. These experimental protocols were approved by the Ethics Committee of the First Affiliated Hospital of Nanchang University.

Human PBMCs or LILs were prepared and stained with conjugated monoclonal antibodies (mAbs). Abs against the following proteins were used: anti-CD3 (SK7), anti-CD16 (CB16), anti-CD56 (CMSSB), anti-CD38 (HB7), anti-CD69 (FN50), anti-FasL (NOK-1), anti-NKG2D (1D11), anti-CD107a (eBioH4A3), anti-perforin (dG9), anti-IFN-γ (4S.B3), mouse IgG1 (P3.6.2.8.1), mouse IgG2a (eBM2a), anti-mouse IgG2a (m2a-15F8) (eBioscience) and anti-TCRγ/δ (B1), anti-GranzymA(GrA) (CB9) (Biolegend) and anti-Vδ2 (B6), anti-Granulysin (RB1), anti-GranzymB(GrB) (GB11) (BD PharMingen) and anti-NKG2A (131411) (R&D) and anti-Vδ1 (TS8.2) (Thermo Scientific) and anti-IFNAR2 (MMHAR-2) (PBL Assay Science). Data were collected on a BD FACSCanto^TM II cytometer and analyzed using FlowJo analysis software 7.6.1 (Tristar).