Pharm lyse lysing buffer

Monocyte subpopulations were identified with flow cytometry using the lysis-no-wash strategy (BD Pharm Lyse lysing buffer, Becton Dickinson) on fresh EDTA blood. A total of 100 μl of EDTA blood was stained by monoclonal antibodies (CD16 FITC NKP15 Becton Dickinson, CD14 PE RMO52 Beckman Coulter, HLA-DR Immu357 PC5.5 Beckman Coulter, and CD45 PC7 J33 Beckman Coulter). Surface expression was assessed using FC500 and CytoFLEX flow cytometer (Beckman Coulter) and analyzed with Kaluza software version 2.1 (Beckman Coulter). The applied gating strategy was in short; monocytes were selected in the SSC/CD45+ plot, gated to SSC/HLA-DR + plot, identifying monocytes as CD45+ HLA-DR + cells with monocyte scatter properties. Exclusion of lymphocytes, and natural killer cells was performed by excluding CD45+ HLA-DR+ CD14– CD16– cells. In the CD14/CD16 plot, the percentages of gated monocyte subsets (classical CD14++CD16−), intermediate (CD14++CD16+), and nonclassical monocytes (CD14+CD16++) were used for analyses. Identification of monocytes subsets follows current recommendations (21 (link)).

Monocyte subpopulations, consisting of CD14⁺⁺CD16⁻ classical monocytes, CD14⁺⁺CD16⁺ intermediate monocytes, and CD14⁺CD16⁺ non-classical monocytes, were identified with the FC500 flow cytometry (Beckman Coulter, Brea, USA) using the lysis-no-wash strategy (BD Pharm Lyse lysing buffer, Becton Dickinson) with 100 μl fresh EDTA blood. Cells were stained by monoclonal antibodies (CD16 FITC Leu11a, Becton&Dickinson; CD14 ECD RM052, Beckman-Coulter; CD45 PC5 J.33, Beckman-Coulter) and subsequently analyzed with Kaluza software version 1.5a (Beckman Coulter).
The full gating strategy is displayed in Supplementary Figure 1. In short, monocytes were gated in SSC/CD45⁺ plot, identifying monocytes as CD45⁺ cells with monocyte scatter properties. Exclusion of lymphocytes and natural killer cells was performed by excluding CD14/CD16 negative cells. Percentages of monocyte subsets (CD14⁺⁺CD16⁻, CD14⁺⁺CD16⁺, and CD14⁺CD16⁺) were identified in the CD14/CD16 plot. For determination of the gates setting, the fluorescence minus one method was applied. Identification of monocyte subsets follows current international recommendations (20 (link), 21 (link)).

BALB/c mice were sacrificed via neck dislocation after anesthesia. Liver perfusion in the mice was performed using 1 × Dulbecco’s phosphate-buffered saline (DPBS) (without Ca²⁺ and Mg²⁺) via the portal vein. Harvested livers were washed with PBS. The tissue was quickly minced with scissors and placed in 50-ml conical tubes. Tissues were dissociated into single cell suspensions using an Ultra Turrax^® Tube Disperser (IKA, Königswinter, Germany). Lymphocytes were separated using a 40% Percoll solution and gradient centrifugation. The supernatant and lipid layer were discarded, and the cells were washed twice with DPBS. Red blood cells were lysed using BD Pharm Lyse™ Lysing Buffer (Becton Dickinson and Company, Franklin Lakes, NJ, USA), leaving nonparenchymal cells. The spleen was removed and ground into a cell suspension, which was filtered into a single-cell suspension using a 70-μm cell sieve (Becton Dickinson). Red blood cells were lysed, and the remaining cells were washed twice with 1× DPBS to obtain splenic lymphocytes.