Erythrocyte lysis buffer

Blood samples were collected in tubes with EDTA anticoagulant. Whole blood was stained with anti-human CD45 phycoerythrin-cyanine5 (CD45-PE-Cy5; eBioscience, USA) and anti-human CD64 phycoerythrin (CD64-PE; Beckman Coulter, USA). After staining, red blood cells were lysed with Erythrocyte Lysis Buffer (BD Biosciences, USA) to obtain peripheral blood mononuclear cells (PBMC). PBMC stained with fluorescent antibodies were examined by flow cytometry (Cytomics™ FC 500, Beckman Coulter, USA). The data were analyzed by flow cytometry analysis software (FlowJo 7.6, LLC, USA). The scatter plot was drawn with side scatter (SSC) and CD45-PE-Cy5, and neutrophils and lymphocytes were gated to acquire their CD64 MFI. The CD64 index was determined by the ratio of the CD64 MFI of granulocytes to that of lymphocytes.

Anti-human monoclonal antibodies (CD3-APC, CD4-PerCP CD25-FITC and CD28-PE) were purchased from BD Pharmingen Company (San Diego, CA, USA). Erythrocyte lysis buffer was purchased from BD Biosciences Company (San Diego, CA, USA). MTB drug-sensitive Roche medium (Roche, USA) was used.
Centrifuges (Jouan, France), an electronic balance (Eppendorff, Germany), a TB culture apparatus (BACTEC MGIT-960) and BBL MGIL culture tubes (BD Company, USA), a flow cytometer (BD Company, USA) and Philips MX4000 Dual Spiral CT Instrument (Philips Corporation, USA) were used.

Spleens were placed in ice cold, 4°C PBS and gently ground between frosted slides to produce a single-cell suspension. The suspension was centrifuged at 400×g for 10 min and the pellet was resuspended in PBS. Red blood cells were lysed with erythrocyte lysis buffer (BD Pharmingen, San Diego, CA) and the remaining cells were washed with PBS by centrifugation at 400×g for 10 min. Cell viability was consistently >95%, as determined using trypan blue exclusion procedure. CD4⁺ or CD8⁺ cells were purified by positive selection using CD4 or CD8 microbeads (>95% purity) obtained from Miltenyi Biotec (Bergisch Gladbach, Germany). For cytokine production, purified CD4⁺ lymphocytes were cultured on plates coated with antibodies to 1 µg/ml of CD3 (145-2C11; BD Pharmingen) and 1 µg/ml of CD28 (37.51; BD Pharmingen) in RPMI 1640 medium (Invitrogen, Carlsbad, CA) with 10% heat-inactivated FBS (Invitrogen) at 37°C, 95% humidity, and 5% CO₂ for 24 h. After incubation, the cell-free suspension was collected and stored at −80°C until further analysis.

These experiments were carried out in accordance with the protocol approved by the Committee on Animal Research at Nara Institute of Science and Technology and the RIKEN Animal Experiment Committee. Subcutaneous fat tissues from three- to four-month-old ICR male mice (n = 3 per sample, two biological replicate samples) were minced into small pieces and incubated with 0.4 U/mL collagenase NB4G (Serva) at 37 °C for 35 min in a shaking water bath. The digested solution was sequentially filtered through 100- and 40-μm cell strainers (Corning), followed by centrifugation at 250×g for 5 min to remove mature adipocytes. The pellet was treated with erythrocyte lysis buffer (BD Biosciences) and centrifuged at 180×g for 5 min. The nucleated cells were suspended in HBSS with 0.1% BSA, filtered through a 20-μm cell strainer (pluriSelect), and then kept on ice (Cell solution A). The cell aggregates that did not pass through the 20-μm strainer were further treated with Accutase (Thermo Fisher Scientific) at 37 °C for 15 min to dissociate them into single cells, centrifuged at 180×g for 5 min, and suspended in HBSS with 0.1% BSA (Cell solution B). Cell solutions A and B were mixed and again filtered through the 20-μm cell strainer, followed by centrifugation at 180×g for 5 min. The pellet was resuspended with HBSS with 0.1% BSA, stained with PI, and used for single-cell analysis.