Cd8 apc antibodies

Autologous T cells were co-cultured with matured DCs for 5 days at a 10:1 ratio, after being submitted to a staining protocol with carboxyfluorescein succinimidyl ester (CFSE). All co-cultures were carried out in U-bottomed 96-well plates in a final volume of 200 µL of RPMI medium. At the end of the co-culture period, cells were stained with fluorescence-conjugated antibodies, namely CD4-PerCP/Cy5.5 and CD8-APC antibodies (BioLegend) in order to evaluate the percentage of positive T cell proliferation. Type 1 T cells (Th1), type 2 T cells (Th2), and regulatory T cells (Treg) subsets were also evaluated through flow cytometry after the co-culture period with DCs for 5 days. The autologous T cells were stained using anti-CD4-PerCP/Cy5.5, anti-CD8-APC, anti-CD25-APC, anti-forkhead-box-P3 (FoxP3)-FITC, anti-GATA-binding protein 3 (GATA3)-FITC, and anti-T-box protein expressed in T cells (T-bet)-PE (Biolegend). As some markers are intracellular, the Cyto-Fast™ Fix/Perm Buffer Set (BioLegend), a fixation and cell permeabilization kit, was used for the intracellular staining, according to the manufacturer’s instructions. Data were analysed with GraphPad Prism version 8 (GraphPad Software, San Diego, CA, USA) and the results are presented as a percentage of positive cells (%) after subtraction of isotype control values.

Amino-terminated monomethyl poly(ethylene glycol) (mPEG-NH₂, M_n = 2000) was bought from Jemkem Inc., China. Tetrahydrofuran (THF), N,N-dimethylformamide (DMF) and toluene were refluxed with CaH₂ and distilled under N₂ before use. γ-Ethyl-L-glutamate N-carboxyanhydride (ELG NCA) was prepared according to our previous method [37 (link)]. All the other chemical reagents were bought from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China), China and used as obtained.
InvivoMab anti-mouse PD-1 (CD279) and invivoMab anti-mouse CTLA-4 (CD152) were purchased from Bioxcell Inc. (West Lebanon, NH, USA). Dox was purchased from Beijing HVSF United Chemical Materials Co., Ltd. (Beijing, China). CD3-FITC, CD4-PE and CD8-APC antibodies were bought from Biolegend Inc (San Diego, CA, USA). Treg cell test kit was bought from Ebioscience Inc. (San Diego, CA, USA). ELISA kits for the detection of IL-2, TNF-α and IFN-γ were obtained from Shanghai Lengton Bioscience Co., Ltd. (Shanghai, China).

The anti-CD4 monoclonal antibody GK1.5 (Cell Culture Company, Minneapolis, MN) was used to deplete CD4⁺ T cells. Mice received the indicated dose of GK1.5 intraperitoneally in 100 μl PBS. Control mice received 100 μl intraperitoneal PBS. CD4⁺ T cell counts in peripheral blood were assessed by performing cheek bleeds in uninfected BALB/c mice. Four mice per dosage group were studied, so two mice per group were assessed each week and 2 weeks elapsed between blood collections from the same mice. For each sample, 100 μl whole blood was obtained and divided into technical duplicates for staining with CD3-PE (1:200 dilution, Cat #100308), CD4-PerCP-Cyanine5.5 (1:200 dilution, Cat #116012), and CD8-APC antibodies (1:200 dilution, Cat #100712) (BioLegend, San Diego, CA), followed by red blood cell lysis and fixation with 1 ml 1× RBC Lysis/Fixation Solution (BioLegend). Cells were then centrifuged at 350 x g for 5 min after which the pellets were resuspended with 275 μl FACS buffer and 25 μl of CountBright Absolute Counting Beads. Cells were analyzed on a 5-laser Bio-Rad ZE5 flow cytometer.