Flow cytometry analysis was performed to evaluate CD4+ and CD8+ T cell depletion and ZIKV-specific CD8+ T cell responses in the challenged mice (Guerrero et al., 1986 (link); Zhang et al., 2016 (link)). For analysis of CD4+ and CD8+ depletion in whole blood and splenocytes, peripheral blood cells and splenocytes were treated with 1 × Red Blood Cell Lysis Buffer (Biolegend), and stained with FITC-anti-mouse CD4 or PerCP-Cy5.5-anti-mouse CD8 antibody (BD Biosciences), followed by flow cytometry analysis using BD LSRFortessa 4 system. For analysis of ZIKV-specific CD8+ T cell responses, the above-treated splenocytes (2 × 106 cells/well) were incubated with ZIKV NS3 overlapping peptides (0.25 nM/peptide, final concentration 5 μg/ml) in the presence of 5 μg/ml brefeldin A (Biolegend), and cultured at 37°C for 5 h. After stimulation, the cells were washed with PBS and stained for surface marker using PerCP/Cy5.5 anti-mouse CD8a. After fixation and permeabilization, the cells were stained for intracellular markers using FITC-anti-mouse IL-2, PE-anti-mouse IFN-γ, and Brilliant Violet 421-anti-mouse TNF-α antibodies (BD Biosciences), followed by analysis using flow cytometry as described above.
Fitc anti mouse il 2
Manufactured by BD
FITC-anti-mouse IL-2 is a fluorescently-labeled antibody that binds to the interleukin-2 (IL-2) protein in mouse samples. It can be used to detect and quantify IL-2 levels in various applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti mouse ifn γ pe, by BD (4 mentions)
Red blood cell lysis buffer, by BioLegend (2 mentions)
Lsrfortessa 4 system, by BD (2 mentions)
Fitc anti mouse cd4 or percp cy5.5 anti mouse cd8 antibody, by BD (2 mentions)
Brilliant violet 421 anti mouse tnf α antibodies, by BD (2 mentions)
Brefeldin a, by BioLegend (2 mentions)
Facsdiva software v6, by BD (2 mentions)
Golgiplug containing brefeldin a, by BD (2 mentions)
Ionomycin, by Merck Group (2 mentions)
Facscalibur, by BD (2 mentions)
Phorbol myristate acetate (pma), by Merck Group (2 mentions)
Cytofix cytoperm plus kit, by BD (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
4 protocols using fitc anti mouse il 2
1
Characterizing ZIKV-specific CD8+ T cells
2
Characterizing ZIKV-specific CD8+ T cells
3
Characterizing T Cell Responses to MERS-CoV and SARS-CoV
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
T cell responses in immunized mice were detected by intracellular cytokine staining followed by flow cytometry analysis as previously described [12] (link), [26] (link). Briefly, splenocytes (2 × 106) were stimulated with or without MERS-CoV S1 protein or SARS-CoV RBD protein (as the negative control) for 3 days at 37 °C with 5% CO2 in the presence of GolgiPlug™ containing brefeldin A (1 μl/ml; BD Biosciences, San Jose, CA). Phorbol myristate acetate (PMA, 20 ng/ml) and ionomycin (2 μg/ml) (Sigma–Aldrich, St. Louis, MO) were used as positive controls. The cells were fixed using a Cytofix/Cytoperm™ Plus Kit in accordance with the manufacturer's protocol (BD Biosciences) and then stained directly with conjugated anti-mouse-CD4 (APC), anti-mouse-CD8 (P-Cy5-5), anti-mouse-IL-2 (FITC), and anti-mouse-IFN-γ (PE) (BD Biosciences) for 30 min at 4 °C. Appropriate isotype-matched controls for cytokines were included in each staining. The stained cells were analyzed using flow cytometry (FACSCalibur; BD Biosciences), and the data were evaluated by FACSDiva software v.6.1.2 (BD Biosciences).
4
Analyzing T Cell Responses in Immunized Mice
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
T cell responses in immunized mice were detected by intracellular cytokine staining followed by flow cytometry analysis as previously described with some modifications.34 (link) Briefly, splenocytes (2×106) were stimulated with or without MERS-CoV S1-His protein. Phorbol myristate acetate (20 ng/ml) and ionomycin (2 µg/ml) (Sigma-Aldrich, St Louis, MO, USA) were used as positive controls. Cells with stimulatory agents were incubated for 6 h at 37 °C with 5% CO2 in the presence of GolgiPlug containing brefeldin A (1 µl/ml; BD Biosciences, San Jose, CA, USA). The cells were stained with conjugated anti-mouse-CD4 (APC) and anti-mouse-CD8 (P-Cy5-5) for 30 min at 4 °C. After washes, the cells were fixed using a Cytofix/Cytoperm Kit in accordance with the manufacturer's protocol (BD Biosciences) and then stained with anti-mouse-IL-2 (FITC) and anti-mouse-IFN-γ (PE) (BD Biosciences) for 30 min at 4 °C. Appropriate isotype-matched controls were included in each staining. The stained cells were analyzed using FACS Calibur (BD Biosciences) and FACSDiva software v.6.1.2 (BD Biosciences).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!