Pe conjugated anti cd8

For surface marker staining, cells were stained in PBS supplemented with 1% FBS for 30 min on ice with the following antibodies (PerCP‐conjugated anti‐CD3, BV421‐conjugated anti‐CD4, PE‐conjugated anti‐CD8, BV570‐conjugated anti‐CD45, BV421‐conjugated anti‐F4/80, BV650‐conjugated anti‐CD86, APC‐conjugated anti‐CD206, PE‐Cy7‐conjugated anti‐CD25, FITC‐conjugated anti‐γδ TCR, APC‐Cy7‐conjugated anti‐CD11b, PE‐conjugated anti‐Ly‐6G, FITC‐conjugated anti‐CD49b, PE‐conjugated anti‐CD19, PE‐conjugated anti‐ST2 antibodies, and APC‐conjugated anti‐NKp46: BD Biosciences, San Jose, CA, USA). After fixation and permeabilization, staining with an APC‐conjugated anti‐FOXP3 antibody (eBioscience) was performed according to the manufacturer's instructions. For IL‐33 staining of primary mouse hepatocytes, cells were stained using a biotinylated anti‐IL‐33 monoclonal antibody (Enzo Life Biosciences, Raamsdonksveer, The Netherlands) and PE‐Cy7‐labeled streptavidin (BD Pharmingen, San Diego, CA, USA). All the flow cytometric data were analyzed and plotted using FlowJo software (TreeStar, Ashland, OR, USA).

FITC-conjugated anti-CD44, PerCP-conjugated anti-CD3, PE-conjugated anti-CD8, FITC-conjugated anti-Granzyme A, anti-Granzyme B, anti-perforin and anti-IFN-γ were all purchased from BD Pharmingen. PE-conjugated anti-CD133 was purchased from eBiosciences. For cell surface staining, SMMC7721 and HepG2 cells were resuspended in 100μl staining buffer containing 10% FBS and put on ice for 20 min to block Fc receptors, then incubated with FITC-conjugated anti-CD44and PE-conjugated anti-CD133or isotype control for 30 min. The cells were then washed with 1ml ice-cold staining buffer for 2 times and centrifuged (300g) at 4°C for 5 min. The collected cells were suspended in 500μl staining buffer solution. PBMCs were incubated with SMMC7721 cells for 5 hours with brefeldin A at a final concentration of 10μg/ml, then collected and washed. As discussed above, PerCP-conjugated anti-CD3 and PE-conjugated anti-CD8 were used for surface staining. After that, cells were fixed and permeabilized, and then intracellular staining of FITC-conjugated anti-GranzymeA, Granzyme B, perforin and IFN-γ was performed. All samples after staining were evaluated using BD FACS Canto II with Diva software and analyzed using Flowjo 7.6.5.

TILs and splenocytes were superficially stained with FITC-conjugated anti-CD3 (clone: 145-2C11, BD Bioscience, US), anti-CD25 (clone: 7D4, BD Bioscience, US), PE-Vio770-conjugated anti-CD4 (clone: REA604, Miltenyi Biotec, Germany), and PE-conjugated anti-CD8 (clone: 53–6.7, BD Bioscience, US) antibodies. The concentration of each conjugated antibody was one microgram per 10⁵ cells in PBS containing 2% fetal bovine serum (FBS). Following incubation at 4 °C for 30 min, the excessive antibodies were removed by washing the cell suspensions. For intracellular staining of FOXP3, cells were fixed and permeabilized using True Nuclear Transcription Factor Buffer Set (BioLegend, San Diego, CA), and PE-conjugated anti-FOXP3 antibody (clone: MF23, BD Biosciences, CA) was added to cells according to the manufacturer protocol. All analyses were carried out on live cells using Zombie NIR Fixable Viability Kit (BioLegend, San Diego, CA). Gating strategies were based on unstained and fluorescent minus one (FMO) controls. Flow cytometry analysis was conducted using an Attune NXT flow cytometer (Invitrogen, US), and data were analyzed by FlowJo software (Tree Star).

WBM was labeled with PB-conjugated anti-B220, PE-conjugated anti-CD4, PE-conjugated anti-CD8, BUV395 conjugated anti-TER 119, APC-conjugated anti-GR1 (BD Biosciences), and Alexa Fluor 488 conjugated anti-CD11b (BioLegend) antibodies. For the B220 double sort discard population, B220 positive (B220 +) cells were isolated by FACS. This primary sorted B220 + population was centrifuged at 1300 rpm for 10 min at 4 °C, the pellet re-suspended in PBS and subjected to reanalysis using the same gating schema as the primary sort. Two populations of cells on the re-analysis, those persistently positive for the B220 and those negative for the B220 (the B220 double sort discard population), were analyzed for the presence or absence of the remaining Lin + markers by flow cytometry. GR1 + cells isolated on primary sort were similarly subjected to double sorting and Lin + marker analysis as above. For stem cell marker analysis, WBM was incubated with APC-conjugated antibodies against either GR1 or B220 (BD Biosciences), and FITC-labeled anti-c-Kit (BD Biosciences), PB-labeled anti- Sca-1 (BioLegend) and PE-labeled anti-CD150 (BioLegend) antibodies. Primary sorted GR1 + cells or B220 + cells were then re-analyzed and percent stem cell marker positive cells in the different cellular populations was determined by flow cytometry.

The following anti-mouse antibodies were obtained from BD PharMingen: PerCP-conjugated anti-CD3e (553067, 1 : 100), PE-conjugated anti-CD8 (557654, 1 : 100), PE-Cy7-conjugated anti-CD8 (552877, 1 : 100), APC-Cy7-conjugated anti-CD8 (557654, 1 : 100), PE-conjugated anti-CD90.1 (561404, 1 : 100), APC-conjugated anti-CD90.1 (557266, 1 : 100), PE-Cy7-conjugated anti-CD62L (560516, 1 : 100), APC-Cy7-conjugated CD62L (560514, 1 : 100), and APC-conjugated anti-IFN-γ (554413, 1 : 100). Biolegend: PE-conjugated anti-CD44 (103008, 1 : 100), PE-Cy7-conjugated anti-CD44 (103030, 1 : 100), PE-Cy7-conjugated anti-CD69 (104512, 1 : 100), and APC-conjugated anti-CD103 (121414, 1 : 100). Flow cytometry data were acquired with a FACS Canto II flow cytometer (BD Biosciences) and data were analyzed with Flowjo software (Tree Star).