Anti mouse cd3 apc

To determine the expression of CD8 + CD28-T cells in mice, FCM analysis was performed on isolated spleen cells using anti-mouse CD3, CD8, and CD28 monoclonal antibodies. The spleens were minced mechanically and lysed in a lymphocyte separation medium. The isolated spleen cells were washed and resuspended in PBS. Anti-mouse CD3 APC, anti-mouse CD8 FITC, and anti-mouse CD28 PE (eBioscience, San Diego, USA) were mixed at 4 0 C for 10 min in the dark. And then, the cell suspension was analyzed using FCM.

Mice spleens were mechanically minced. After lysing with lymphocyte separation medium, cells were resuspended in PBS. With 10-min incubation of anti-mouse CD3 APC, anti-mouse CD8 FITC, and anti-mouse CD28 PE (eBioscience, San Diego, USA) at 4 °C in the dark, the cell suspension was analyzed by FCM.

Spleen immune cells were isolated from 129 mice (n = 6) and Dok-3^−/− mice (n = 6). Mice spleens were isolated from the body and grinded by the copper grid. The immune cells of spleen were obtained after the 10 min incubation with red blood cell lysis buffer. The cells were stained with anti-mouse Tim-3-PE (ebioscience), anti-mouse CD3-APC (ebioscience) or anti-mouse CD3-FITC (ebioscience) which was used in DC cells marking, anti-mouse CD4-FITC (ebioscience), anti-mouse CD8-FITC (ebioscience), anti-mouse CD11b-Pe-cy-7 (ebioscience), anti-mouse NK1.1-PerCP-5.5 (ebioscience), anti-mouse CD11c-APC (ebioscience), anti-mouse Gr-1-FICT (ebioscience) for 30 min. At least 10,000 cells were analyzed by a FACSAriaII. Cells were gated based on their forward and side scatter properties. The cells’ gated strategy is shown in Additional file 1: Figure S1.

Peripheral blood was collected by retro-orbital bleeding, and bone marrow cells were isolated from the femurs and tibias of leukemic mice. Flow cytometry and cell cycle analyses were performed as we described previously [47 (link)]. Briefly, leukemia cells were stained with anti-mouse Mac-1-APC, anti-mouse Gr-1-PE, anti-mouse CD3-APC, anti-mouse B220-PE or anti-mouse c-Kit-PE monoclonal antibodies (eBioscience). The cell cycle stages were evaluated with either Ki-67/7-AAD staining (BD Pharmingen) or a 5-bromo-2′-deoxyuridine (BrdU) incorporation assay. For the analysis of apoptosis, leukemia cells were stained with PE-conjugated anti-Annexin V and 7-AAD (BD Pharmingen) according to the manufacturer's protocol. For the measurement of ROS, the cells were incubated with 1 μM 5-(and-6)-carboxy-2′,7′-dichlorofluorescein diacetate (carboxy-DCFDA, Invitrogen) for 30 minutes at 37°C, followed by flow cytometric analysis. For the examination of the BrdU incorporation assay, leukemic mice were subjected to three intraperitoneal injections of BrdU (Sigma; 3 mg/24 hours) in PBS. The BM cells were fixed, permeabilized and denatured, followed by antibody staining with anti–BrdU-APC according to the manufacturer's instructions (BD Pharmingen).

H₂O₂ and CT were obtained from Sigma-Aldrich (St. Louis, MI, USA). Anti-mouse CD3-APC, CD4-FITC, CD8-PE, CD11c-eFluor 450, MHCII-APC, CD5-FITC and B220-PerCP-eFluor™ 710 antibodies were bought from eBioscience (San Diego, CA, USA). Horseradish Peroxidase (HRP)-conjugated goat anti-mouse IgA, HRP-conjugated goat anti-mouse IgG, HRP-conjugated goat anti-mouse IgG1 and HRP-conjugated goat anti-mouse IgG2a were obtained from Southern Biotech (Birmingham, AL, USA). RNA-easy Isolation was purchased from Vazyme Biotech Co., Ltd. (Nanjing, Jiangsu Province, China). SYBR qPCR Master Mix for quantitative real-time PCR (qRT-PCR) was bought from Yeasen Biotechnology Co., Ltd. (Shanghai, China). TMB was purchased from Beyotime Biotechnology (Shanghai, China). Red blood cell lysis buffer was purchased from Solarbio Science & Technology Co., Ltd. (Beijing, China). Silane coupling agent (KH550) and ZnONPs (purity 99.9%) were the products of Sinopharm Chemical Reagent Ltd. (Shanghai, China). The purified AMP was prepared in our laboratory (Yangzhou, Jiangsu Province, China). H₂SO₄, alcohol and HCl were obtained from Hushi Laboratory Equipment Co., Ltd. (Shanghai, China). Elisa plate was bought from cellpro biotechnology Co., Ltd. (Suzhou, Jiangsu Province, China).

The percentages of CD4⁺ and CD8⁺ T lymphocytes subsets and the production of cytokine in the splenocytes of immunized mice were determined using flow cytometry techniques as described previously (Zhang et al., 2015b (link); Wang S. et al., 2017 (link)). Briefly, splenocytes (2 × 10⁶cells/ml) of vaccinated mice were stimulated with G10E peptides (10 μg/mL) for 72 h. Then the suspensions were stained with anti-mouse CD3-APC, anti- mouse CD4-FITC and anti-mouse CD8-PE (eBiosciences, USA) for 30 min at 4 °C in the dark.
Splenocytes (2 × 10⁶ cells/ml) were also stimulated with G10E peptides for 72 h in the presence of Cell Stimulation Cocktail (eBiosciences, USA) containing Phorbol myristate acetate (PMA, 20 ng/ml), Ionomycin (2 μg/ml), Brefeldin A (1 μg/ml), and Monensin (1 μg/ml) to inhibit the secretion of cytokine into the extracellular space. The cells were fixed using an Intracellular Fixation & Permeabilization Buffer Set Kit in accordance with the manufacturer's protocol (eBiosciences, USA) and then stained directly with anti-mouse CD4-FITC, anti-mouse CD8-PE, anti-mouse IL-2 (APC), anti-mouse IFN-γ (PerCP-Cyanine 5.5), anti-mouse IL-4 (APC), and anti-mouse IL-10 (PerCP-Cyanine5.5) (eBiosciences, USA) for 30 min at 4°C. All these cell population were analyzed by Cytoflex S Flow Cytometer (Beckman Coulter, USA) and the data were analyzed by CytExpert software.