EAE mice were sacrificed on day 20, and mononuclear cells from spleens were separated by using density gradient centrifugation. The cells were co-cultured in 24-well plate (1×106 cells/well) with MOG35–55 or MOG40–54 peptides (25 μg/mL) for 16 h at 37°C under 5% CO2 and humidified conditions, followed by the addition of protein transport inhibitor Brefeldin A (Multi Sciences, Shanghai, China) for another 5 h. Cells were harvested and blocked with anti-mouse CD16/32 (2.4G2; eBiosciences) for 20 min and then stained with FITC-anti-mouse CD4 and APC-anti-mouse CD8a (eBiosciences) for 30 min at 4°C. After that, the cells were incubated with PE-anti-mouse interferon (IFN)-γ (XMG1.2) or PE-anti-mouse IL-17A (eBio17B7) (eBiosciences) for 30 min at 4°C after fixation/permeabilization and analyzed by flow cytometry. The gating strategy for each figure of flow cytometry was described in Supplementary materials.
Apc anti mouse cd8a
Manufactured by Thermo Fisher Scientific
Sourced in United States
APC anti-mouse CD8a is a flow cytometry reagent used to detect and identify CD8a-positive cells. CD8a is a glycoprotein expressed on the surface of certain T cells and natural killer cells. This reagent is conjugated with Allophycocyanin (APC), a fluorescent dye, which allows for the detection and quantification of CD8a-positive cells by flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Anti mouse cd4 fitc, by Thermo Fisher Scientific (2 mentions)
Anti mouse cd16 32 2.4g2, by Thermo Fisher Scientific (1 mentions)
Brefeldin a, by MultiSciences Biotech (1 mentions)
Apc cy7 anti mouse cd3, by BD (1 mentions)
Brefeldin a, by Thermo Fisher Scientific (1 mentions)
Anti mouse ifn γ pe, by BD (1 mentions)
Anti mouse cd4 pe cy7, by Thermo Fisher Scientific (1 mentions)
Live dead fixable aqua dead cell stain kit, by Thermo Fisher Scientific (1 mentions)
Facscanto 2, by BD (1 mentions)
Monensin, by BioLegend (1 mentions)
Percp anti mouse cd11b, by BioLegend (1 mentions)
Bv510 anti mouse cd45 antibody, by BioLegend (1 mentions)
Anti mouse cd3 fitc, by Thermo Fisher Scientific (1 mentions)
Bv421 anti mouse nk1, by BioLegend (1 mentions)
Anti mouse f4 80 apc, by Thermo Fisher Scientific (1 mentions)
Facsaria 2 cell sorter, by BD (1 mentions)
Anti mouse cd206 bv421, by BioLegend (1 mentions)
Facsaria 2 flow cytometer, by BD (1 mentions)
Facsversetm flow cytometer, by BD (1 mentions)
Ab32529, by Abcam (1 mentions)
6 protocols using apc anti mouse cd8a
1
Evaluation of MOG-specific T-cells in EAE mice
2
Multiparametric Flow Cytometry for Evaluating Cytokine Responses
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Intracellular cytokine staining (ICS) was performed for TNFα and IFNγ. Peripheral blood cells (collected 7 days after each booster dose) and lung leukocytes (collected after the final booster dose) were stimulated overnight with 1 μg per peptide per well of Spike-derived overlapping peptides at 37°C, 5% CO2 in the presence of brefeldin A (Invitrogen, catalog no. 00-4506-15) and monensin (BioLegend, catalog no. 420701). Cells were stained with the following antibodies: PE anti-mouse IFNγ (BD, catalog no. 554412), fluorescein isothiocyanate anti-mouse TNFα (BD, catalog no. 554418), APC-Cy7 anti-mouse CD3 (BD, catalog no. 560590), PE-Cy7 anti-mouse CD4+ (Invitrogen, catalog no. 25-0041-82), and APC anti-mouse CD8a (eBioscience, catalog no. 17-0081-83). PMA (50 ng/ml) and ionomycin (1 μM) were used as positive controls, and complete medium only was used as the negative control. Cells were permeabilized and fixed (Invitrogen, catalog no. 00-5523-00). A LIVE/DEAD fixable (aqua) dead cell stain kit (Invitrogen, catalog no. L34966) was used to evaluate viability of the cells during flow cytometry. Sample acquisition was performed on FACSCanto II (BD) and data were analyzed with FlowJo V10 software (BD).
3
Multicolor Flow Cytometry Immunophenotyping
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Cells were incubated with Fc block antibody (#14-0161-82, eBioscience, 1:100) for 30 min at 4 °C to prevent nonspecific binding. Cell surface labeling was conducted with fluorescently conjugated antibodies for 30 min at 4°C. The following antibodies were used: BV510 anti-mouse CD45 antibody (#103137, Biolegend, 1:25), PerCP anti-mouse CD11b (#101229, Biolegend, 1:50), APC anti-mouse F4/80 (#17-4801-80, eBioscience, 1:25), PE/Cy7 anti-mouse Gr1 (#108415, Biolegend, 1:100), FITC anti-mouse CD3 (#11-0032-82, eBioscience, 1:50), PE/Cy7 anti-mouse CD19 (#25-0193-81, eBioscience, 1:50), BV421 anti-mouse NK1.1 (#108741, Biolegend, 1:20), PE anti-mouse CD36 (#562702, BD Biosciences, 1:100), PE anti-mouse CD4 (#4329629, Invitrogen, 1:200), APC anti-mouse CD8a (#17-0081-81, eBioscience, 1:200), BV421 anti-mouse CD206 (#141717, Biolegend, 1:25), FITC anti-mouse CD80 (#FITC-65076, Proteintech, 1:200), FITC anti-mouse GzmB (#372206, Biolegend, 1:25), FE anti-mouse IFNγ (#PE-65153, Proteintech, 1:50). Fluorescence data were collected using a FACSAria II flow cytometer (BD Biosciences, USA) and analyzed employing a FlowJo software. The single cell population was separated with a BD FACSAria II Cell Sorter.
4
Profiling T-cell Responses in Mycobacterial Infection
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Lung and spleen tissues were harvested at various time points after the challenge with M. bovis. The tissues were transferred to sterilized petri dishes (50 mm) containing an RPMI 1640 medium, 10% FBS, 1% penicillin/streptomycin. Single-cell suspensions from lung and spleen tissue were obtained as described previously [43 (link)]. Single-cell suspensions were stimulated with Early secretory antigenic target-6 (ESAT-6) (5 μg/mL) for 12–18 h at 37 °C. Cell suspensions were then stained with fluorochrome labeled antibodies for anti-CD4 (anti-mouse CD4 FITC), anti-CD8 (Anti-Mouse CD8a APC), and anti-CD3 (Anti-Mouse CD3e PerCP-Cyanine5.5) from eBioscience (San Diego, CA, USA). Cells were analyzed with BD FACSVerseTM flow cytometer (BD Biosciences, San Jose, CA, USA) using FlowJo X software.
5
CD8+ Tem Cell Phenotyping by Flow Cytometry
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We used flow cytometry to isolate and count CD8+ Tem cells. Cells were labeled with the following fluorescently conjugated monoclonal antibodies: anti-mouse CD45-PE (12045181, eBioscience, San Diego, CA, USA), anti-mouse CD8a-APC (17008181, eBioscience), anti-mouse CD44-PE (25044181, eBioscience), and anti-mouse CD62L (104432, BioLegend, San Diego, CA, USA). After the CD8+ Tem cells were sorted by flow cytometry, they were stained with antibodies against mTOR (ab87540, Abcam, Cambridge, MA, USA), interferon γ (IFN-γ) (11731181, eBioscience), T-bet (ab91109, Abcam), S6K (ab32529, Abcam) and Eomes (53-4875-82, eBioscience).
6
Fura-2 AM and ORAI1-CT Calcium Signaling
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Fura-2 AM calcium indicator was purchased form Life Technologies (Carlsbad, CA, USA). Phorbol 12-myristate 12-acetate (PMA), ionomycin, thapsigargin (TG) and isopropyl-ginethiogalactopyranoside (IPTG) were purchased from Sigma Aldrich (St Louis, MO, USA). Tri(2-carboxyethyl)phosphine (TCEP) was obtained from Pierce (Life Technologies). Amylose resin used for MBP pulldown was purchased from New England Biolabs (Ipswich, MA, USA). Ni-NTA resin used for purification of GB1-ORAI1-CT was purchased from Qiagen (Valencia, CA, USA). The mouse monoclonal anti-Flag M2-HRP (A859, Sigma-Aldrich, St. Louis, MO, USA) antibody, the rabbit anti-mCherry polyclonal antibody (NBP2-25157, Novus Biologicals, Littleton, CO, USA), the rabbit anti-Caspase-1 antibody (D7F10, Cell signaling, Danvers, MA, USA) and the rabbit anti-IL-1β antibody (sc-7884, Santa Cruz Biotechnology, Dallas, TX, USA) were used at a 1:1000 dilution. For flow cytometry (FACS) analysis, anti-mouse MHC Class II (I-A/I-E) APC (), anti-mouse IFN gamma PE (), anti-mouse CD86 (B7-2) PE(12–7311), anti-mouse CD197 (CCR7) PE (12–1971), anti-mouse MHC Class I PE (12–9558), anti-mouse CD11c FITC (11–0114), anti-mouse CD4 PerCP-Cyanine5.5 (45–0042), and anti-mouse CD8a APC (17–0081) were purchased from eBioscience. All other reagents were form Sigma-Aldrich unless otherwise indicated.
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