Flow cytometry analysis was performed to detect ZIKV-specific cellular immune responses as previously described [29 (link)]. Briefly, splenocytes (2 × 106 cells/well) were isolated from immunized mice 3 days post-ZIKV challenge, and treated with 1 × Red Blood Cell Lysis Buffer (Biolegend, San Diego, CA, USA), followed by incubation with a ZIKV EDIII peptide mixture (final concentration of 5 μg/mL) (Table 1 ) in the presence of brefeldin A (5 μg/mL, Biolegend), and cultured at 37 °C for 12 h. After stimulation, the cells were washed with PBS, and stained for surface markers using PerCP/Cy5.5 anti-mouse CD8-positive (CD8+), FITC-anti-mouse CD4-positive (CD4+), and AF700 anti-mouse CD45 antibodies (Biolegend). After fixation and permeabilization, the cells were further stained for intracellular markers using PE anti-mouse interferon-gamma (IFN-γ), Brilliant Violet 711TM anti-mouse interleukin 4 (IL-4), and Brilliant Violet 605TM anti-mouse IL-17 antibodies (Biolegend), which were analyzed using a flow cytometer (BD LSRFortessa 4 system).
Lsrfortessa 4 system
Manufactured by BD
Sourced in United States
The BD LSRFortessa 4 system is a multi-color flow cytometer designed for advanced multiparameter analysis. It features four lasers and the capability to detect up to 18 parameters simultaneously. The system is equipped with a high-throughput sample injection system and supports a variety of sample types, including cells, particles, and microorganisms.
Automatically generated - may contain errors
Lab products found in correlation
Brefeldin a, by BioLegend (3 mentions)
Red blood cell lysis buffer, by BioLegend (3 mentions)
Fitc anti mouse cd4 or percp cy5.5 anti mouse cd8 antibody, by BD (2 mentions)
Fitc anti mouse il 2, by BD (2 mentions)
Brilliant violet 421 anti mouse tnf α antibodies, by BD (2 mentions)
Anti mouse ifn γ pe, by BD (2 mentions)
5 protocols using lsrfortessa 4 system
1
Comprehensive Analysis of ZIKV-Specific Immune Responses
2
SARS-CoV-2 RBD-ACE2 Interaction Assay
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Flow cytometry was performed to detect the interaction between the SARS-CoV-2 RBD and ACE2 in the presence of mouse sera. Briefly, human ACE2-expressing HEK293T cells were incubated with recombinant Fc-tagged SARS-CoV-2 RBD (0.1 μg/ml) in the presence or absence of serially diluted mouse sera at room temperature for 1 h. After three washes with PBS (containing 2% FBS), the cells were incubated with FITC-labeled goat anti-human IgG-Fc antibody (1:500) (Sigma-Aldrich) at room temperature for 20 min. After more washes, the cells were fixed with 4% formaldehyde and the fluorescence intensity of the cells was measured using flow cytometry (BD LSRFortessa 4 system).
3
Characterizing ZIKV-specific CD8+ T cells
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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.
4
MERS-RBD Binding Inhibition Assay
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Sera from immunized mice were tested for their ability to inhibit the binding of the recombinant MERS-RBD-Fc protein to the cell-associated hDPP4 receptor in Huh-7 cells using flow cytometry analysis [26 (link)]. Briefly, cells were incubated with the MERS-RBD-Fc protein (5 µg/mL) in the presence or absence of diluted mouse sera (1:50) for 30 min at room temperature. After three washes and staining with FITC-labeled goat anti-human IgG Fc secondary antibody (1:500, Thermo Fisher Scientific, Waltham, MA, USA) for 30 min at room temperature, the cells were measured for fluorescence in a flow cytometer (BD LSRFortessa 4 system). Mean fluorescence intensity (MFI) values of the FITC channel from cells incubated with MERS-RBD-Fc protein in the absence of diluted sera were treated as 100 percentage binding. Inhibition of binding was calculated as the percentage of reduced binding to hDPP4 receptor in the presence of diluted sera from the different immunization groups versus the maximum binding observed in the absence of sera.
5
Characterizing ZIKV-specific CD8+ T cells
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