At 7 dpi, pulmonary single-cell suspension was obtained and labeled using the method described above but with the following labeling antibodies: APC anti-mouse CD11c, FITC anti-mouse MHC Class II, PE anti-mouse NKp46, PE/Cy7 anti-mouse CD19, PerCP/Cy5.5 anti-mouse CD3ε, PE anti-mouse F4/80, PE/Cy7 anti-mouse Ly-6G, PerCP/Cy5.5 anti-mouse Siglec H (all from BioLegend, USA), FITC anti-mouse CD4 and PE anti-mouse CD8a (both from eBioscience).
Anti mouse cd8a pe
Manufactured by Thermo Fisher Scientific
Sourced in United States
Anti-mouse CD8a-PE is a flow cytometry antibody conjugate used to detect and quantify the CD8a receptor on the surface of mouse cells. CD8a is a cell surface glycoprotein expressed primarily on cytotoxic T lymphocytes and is involved in antigen recognition and T cell activation. The PE (phycoerythrin) fluorophore enables detection and analysis of CD8a-positive cells by flow cytometry.
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Lab products found in correlation
Anti mouse cd4 fitc, by Thermo Fisher Scientific (3 mentions)
Anti mouse cd4 apc, by Thermo Fisher Scientific (2 mentions)
Pe anti mouse nkp46, by BioLegend (1 mentions)
Pe cy7 anti mouse cd19, by BioLegend (1 mentions)
Pe cy7 anti mouse ly 6g, by BioLegend (1 mentions)
Percp cy5.5 anti mouse cd3ε, by BioLegend (1 mentions)
Pe anti mouse f4 80, by BioLegend (1 mentions)
Apc anti mouse cd11c, by BioLegend (1 mentions)
Anti mouse cd3 apc, by Thermo Fisher Scientific (1 mentions)
Mouse il 12p70 elisa kit, by Dakewe (1 mentions)
Mouse il 10 elisa kit, by Dakewe (1 mentions)
Anti mouse cd25 pe, by Thermo Fisher Scientific (1 mentions)
Apc anti mouse cd206, by Thermo Fisher Scientific (1 mentions)
Methylthiazoltetrazolium mtt, by Merck Group (1 mentions)
Mouse tnf α elisa kit, by Dakewe (1 mentions)
Accuri c6 equipment, by BD (1 mentions)
Fix perm buffer, by Thermo Fisher Scientific (1 mentions)
Ononin, by Chengdu Pufei De Biotech (1 mentions)
Calycosin, by Chengdu Pufei De Biotech (1 mentions)
Calycosin 7 o β d glucoside, by Chengdu Pufei De Biotech (1 mentions)
6 protocols using anti mouse cd8a pe
1
Pulmonary Immune Cell Phenotyping
2
Comprehensive Immune Profiling of Nanoparticle Formulations
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SF was provided by Shanghai Biochempartner Co., Ltd. (Shanghai, China). Injectable soya lecithin was provided by Shanghai Taiwan Pharmaceutical Co., Ltd. (Shanghai, China). Coumarin-6 (C6) was bought from Aladdin Chemical Co., Ltd. (Shanghai, RPC). DSPE-rhodamine B was purchased from Ruixi Biological Technology Co., Ltd (Xi’an, China). Methylthiazol tetrazolium (MTT) were purchased from Sigma-Aldrich (US). APC anti-mouse CD3, FITC anti-mouse CD4, PE anti-mouse CD8a, PE anti-mouse CD25, and Alexa Fluor® 647 anti-mouse FOXP3 were purchased from eBioscience. Alexa Fluor ® 488 anti-mouse CD86, PerCP/Cy5.5 anti-mouse F4/80, and APC anti-mouse CD206 were bought from eBioscience. Mouse IL-12p70 Elisa kit, Mouse TGF-β1 Elisa kit, Mouse IL-10 Elisa kit, and Mouse TNF-α ELISA kit were purchased from DAKEWE. All other reagents were of analytical grade and obtained commercially.
3
Profiling Immune Cell Populations in Spleen and Tumors
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For the detection of lymphocytes in the spleen and TIL, 2 × 106 cells were used. Labeling was performed for 30 min at 4°C in the dark using the antibodies in a 1 : 10 dilution in staining buffer (2% FBS in PBS) for surface antibodies and Fix-Perm buffer (intracellular fixation permeabilization buffer set, eBioscience, 88-8824-00) for intracellular antibodies. To ensure lymphocyte population analysis, the CD45 label was made with the CD45.2-APC anti-mouse antibody (eBioscience, Clone: 104). For the detection of the CD8+ and CD4+ populations, the anti-mouse CD8a-PE (eBioscience, Clone: 53-6.7) and anti-mouse CD4-FITC (eBioscience, Clone: RM4-5) antibodies were used, respectively. For the detection of CD4+ subpopulations, the cells were fixed and permeabilized with Fix-Perm buffer. The CD4+ Foxp3+ (Treg) population was detected using the anti-mouse/rat Foxp3-PE-Cy5 antibody (eBioscience, Clone: FJK-16s). The CD4+ IFNγ+ (Th1) and CD4+ IL-17A+ (Th17) populations were detected using the IFNγ-PE anti-mouse (eBioscience, Clone: XMG1.2) and IL-17A-PerCP anti-mouse (eBioscience, Clone: TC11-18H10.1) antibodies, respectively, in lymphocytes previously activated with PMA and Ionomycin. BD Accuri C6 equipment (BD Biosciences, USA) was used for the acquisition of flow cytometry data and FlowJo 7.6.1 software (for the population's analysis).
4
HPLC-Based Phytochemical Analysis
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Astragalosides (I, II, III and IV), calycosin-7-O-β-d-glucoside, ononin, calycosin and formononetin (the purities of all standards were higher than 98% by HPLC analysis) were purchased from Chengdu Pufeide Biotech Co., Ltd. (Chengdu, China). Acetonitrile (ACN) as HPLC grade was purchased from Merck (Darmstadt, Germany). All chemicals used were analytical grade and purchased from Sigma-Aldrich (St. Louis, MO, USA). Milli-Q water prepared from Sigma-Aldrich Co. (Millipore, MA, USA).
Anti-mouse CD3-FITC, anti-mouse CD4-APC, and anti-mouse CD8a-PE were provided by eBioscience (San Diego, CA, USA). Enzyme-linked immunosorbent assay (ELISA) kits IL-2, IL-6 and MCP-1 were supplied by Neobioscience Technology Co., Ltd. (Shenzhen, China). Primary antibody against COX-2 and the secondary antibody were purchased from Cell Signaling Technology (Danvers, MA, United States), all antibodies were diluted with 1:1000. Astragaloside I, Astragaloside II, Astragaloside III, Astragaloside IV (the purities of all standards were higher than 98% by HPLC analysis) were purchased from Chengdu Pefeide Biotech Co., Ltd. (Chengdu, China). Acetonitrile (ACN) purchased from Merck (Darmstadt Germany) is HPLC grade. And other chemicals used were analytical grade and purchased from Sigma-Aldrich (St. Louis, MO, USA).
Anti-mouse CD3-FITC, anti-mouse CD4-APC, and anti-mouse CD8a-PE were provided by eBioscience (San Diego, CA, USA). Enzyme-linked immunosorbent assay (ELISA) kits IL-2, IL-6 and MCP-1 were supplied by Neobioscience Technology Co., Ltd. (Shenzhen, China). Primary antibody against COX-2 and the secondary antibody were purchased from Cell Signaling Technology (Danvers, MA, United States), all antibodies were diluted with 1:1000. Astragaloside I, Astragaloside II, Astragaloside III, Astragaloside IV (the purities of all standards were higher than 98% by HPLC analysis) were purchased from Chengdu Pefeide Biotech Co., Ltd. (Chengdu, China). Acetonitrile (ACN) purchased from Merck (Darmstadt Germany) is HPLC grade. And other chemicals used were analytical grade and purchased from Sigma-Aldrich (St. Louis, MO, USA).
5
Multicolor Flow Cytometry of Mouse Splenocytes
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The collected mouse spleens from the four groups were weighed on an electronic balance and then grinded via 200 mesh screen to obtain single spleen cell suspension. The spleen cell suspension was lysed by RBC Lysis Buffer. Then, the cells (300–400 g) were spun at 4 °C, and the pellets were suspended in the appropriate PBS. For each staining, 1-2 × 109 cells were fluorescently labeled after incubation in a dark room for 30 min at 4 °C with the following antibodies (all from Ebioscience, San Diego, CA, USA): anti-mouse CD3e FITC (no. 11-0031), anti-mouse CD4 APC (no. 17-0041), anti-mouse CD8a PE (no. 12-0081), and anti-mouse CD11c PE-Cyanine7 (no. 25-0114). All samples were detected on a flow cytometer (FACSCalibur; BD Biosciences, CA, USA) and analyzed with FlowJo ver. 7.6 software.
6
Highly Purified CD8+ T Cell Isolation
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CD8þ T cells were highly purified from the spleens of C57BL/6 mice using magnetic bead separation (CD8þ cell isolation kit; Miltenyi Biotec, Bergisch-Gladbach, Germany). In brief, spleens from male C57BL/6 mice were removed and disrupted over wire mesh screens. Red blood cells were lysed in 0.85% NH 4 in Tris-HCl buffer. Isolated CD8þ T cells were incubated with antimouse CD8a PE (eBioscience, San Diego, California) for 30 min at 4°C in the dark. CD8þ T cells were washed three times, resuspended in flow cytometry staining buffer (eBioscience, San Diego, California), and the percentage of CD8þ T cells stained with specific reagents was determined by FACSCalibur and FlowJo software (Tree Star Inc., Ashland, Oregon). The purity of populations was determined by flow cytometric analysis and routinely reached >95%.
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