Fixation permeabilization buffer

A cocktail of conjugated Ab was used to phenotype lung cell suspensions for multi-parameter flow-cytometry (see Supplementary Table S5). Lung cell suspensions were treated with human IgG to block Fc-receptors and stained for surface markers. Cells were then washed and treated with fixation/permeabilization buffer (ThermoFisher). Following permeabilization, cells were stained for intracellular markers, washed, and resuspended in PBS. Cells were collected on a FACS Fortessa and analyzed with BD FACSDiva Software v8.0.1 (BD Biosciences).

Full-spectral flow cytometry was used for immunophenotyping of B lymphocytes, T lymphocytes and NK lymphocytes of the peripheral blood samples (Figure S1). Detailly, the mixed non-bauble violet (BV) antibodies and brilliant staining buffer (Becton, Dickinson and Company, New York, USA) were added to the peripheral blood samples, and incubated for 15 min at room temperature in the dark. Then, lysis buffer (Becton, Dickinson and Company, New York, USA) was added to the samples, and the supernatant was discarded following 10 min of centrifugation. Sediment was washed with fixation/permeabilization buffer (Thermo Fisher Scientific, Massachusetts, USA) followed by centrifugation and incubated with cytoplasmic antibodies for 30 min in the dark. Phosphate buffered saline (Beyotime Biotechnology, Shanghai, China) was used to wash the samples. Finally, the supernatant was removed by centrifugation, and processed in a full-spectrum flow cytometer (Shanghai Xiatai Biotechnology Co., Ltd., Shanghai, China).

Cell suspensions of isolated primary microglia, adult microglia, or BV2 cells were stained with the indicated antibodies or challenged with fluorescent microspheres and then subjected to flow cytometric analysis by using a Beckman Gallios flow cytometer as described before [55 (link)]. For the intracellular staining of Peli1, C/EBPβ, and Aβ, the cells were fixed and permeabilized by fixation/permeabilization buffer (00-5223-56 and 00-5123-43, Thermo Fisher) before staining the specific primary antibodies and followed by fluorescent-labeled secondary antibodies staining, and then detected by flow cytometer.

The following antibodies were used: antihuman CD3-FITC or V500, CD4 PerCP-Cy5.5, CD8 FITC or V450, CD56 PE or BV510, IFN-γ APC, PD-L1 APC, and mIgG1 APC isotype control (all from BD Biosciences). Cell surface staining was performed by incubating cells with mAbs for 30 min at 4°C in FACS staining buffer (BD Biosciences) followed by 2 times washing with phosphate-buffered saline. For intracellular staining, surface stained cells were fixed and permeabilized using fixation/permeabilization buffer (Thermo Fisher Scientific) and stained with anti-IFN-γ mAb according to the manufacturer’s instructions. Samples were resuspended in FACS buffer and acquired using a LSRFortessa flow cytometer (BD Biosciences) with FACSDiva software and analyzed using FlowJo software (FlowJo). For FACS data analysis, doublets were excluded using forward scatter height and width properties and dead cells were excluded by FVD780 (Thermo Fisher Scientific) positive staining.

The infiltrated immune cells from WT and Tpl2-deficient inflamed livers were prepared through 33% Percoll gradient as previously described (6 (link)). The collected liver-infiltrated immune cells or splenic cell suspensions were stained with the indicated antibodies and were subjected to flow cytometry analyses as previously described by using a Beckman Gallios flow cytometer (39 (link)). For the intracellular staining of TNF-α, IFN-γ, and Foxp3, the cells were fixed and permeabilized by fixation/permeabilization buffer (Thermo Fisher) before staining these antibodies, and then detected by flow cytometer. The absolute numbers of splenic and liver-infiltrating immune cells subpopulations were calculated based on their frequencies and the total number of isolated splenic and liver immune cells, and the data were presented as the average numbers of immune cell subpopulations per one spleen or liver of one mouse.