Dxp athenatm v5 b5 r3

For cell signaling detection, cells were cultured in 12-well plates, treated with IFN-g for 24 h, and harvested in 1% FBS/PBS. After treatment with CD16/CD32 Fc block antibody, cells were incubated with anti-PD-L1 or anti-MHC Class I antibodies for 30 min at 4 C. Then cells were washed once with 1% FBS/PBS and resuspended in 200 ml 1% FBS/PBS.
For tissue detection, LLC-exograft tumors were digested with collagenase P (2 mg/ml in PBS, Roche) supplemented with DNase I (5 ml/ml) for 1 h at 37 C. A single cell suspension was obtained in 1% bovine serum albumin/PBS by filter filtration. Cells were incubated with mouse CD16/CD32 Fc block antibody for 10 min at 4 C, followed by incubation with fluorochrome-conjugated antibodies: anti-CD45, anti-CD3, anti-CD8, anti-CD4 and anti-H-2Kb. For intracellular staining, cells were washed in 1% bovine serum albumin /PBS after surface staining, fixed, and permeabilized with Cyto-Fix/Perm buffer set (BD Pharmingen) for anti-GZMB staining, respectively.
All data were acquired immediately using cytometer (DxP AthenaTM V5-B5-R3, Cytek Biosciences) or cytoFLEX (BECKMAN) and further analysed using FlowJo 10.0.

Cells were fixed in 4% paraformaldehyde for 10 minutes at RT, followed by permeabilization with treatment of 0.2% TritonX-100 in PBS for 10 minutes. Cells were then blocked with PBS containing 5% bovine serum albumin for 30 minutes and incubated with anti-IFNGR1, anti-IFNGR2 or anti-p62 antibodies overnight at 4 C. Then cells were incubated with anti-mouse Alexa Fluor 488 dye conjugate or anti-rabbit Alexa Fluor 594 dye conjugate for another 1 h at RT. 4',6-diamidino-2-phenylindole (DAPI) were incubated for 5 minutes at RT for nuclei staining. Cells were imaged using a confocal laser scanning microscopy (Leica SP8-DMIL) and the data were analyzed by Leica LAS AF Lite 3.3.0.
In vitro killing assay OVA-specific CD8 + CD45.1 + T cells were isolated from OT-I [C57BL/6-Tg(TcraTcrb)1100Mjb/J] mouse stain using CD90.2 microbeads (Miltenyi Biotec) and pre-activated with anti-mouse CD3/CD28 (bioxcell) for 48-72 h. LLC-OVA cells with control or Trim35 KO were plated in 96-well plate (1310 5 per well) with activated CD8 + T cells in 1:1 or 2:1 ratio in triplicates. After 6-8 h incubation, both T cells and LLC cells were collected and stained with anti-CD45.1 (eBioscience), anti-CD8a and anti-cleaved caspase3 (BD). The percentage of cleaved caspase3 + CD8 -CD45.1 -cells were analyzed using a cytometer (DxP AthenaTM V5-B5-R3) from Cytek Biosciences and further calculated using FlowJo 10.0.