The cells isolated from spleen tissues of vaccinated mice and controls were abundant in yield. Splenocytes (2.5 × 106) were incubated with 0.1 × 106 PFU live ZIKV FSS13025 for 24 h. BD GolgiPlug (BD Bioscience) was added to block protein transport at the final 5 h of incubation. Cells were stained with antibodies for CD3 (25-0031-81; 10 µg/ml), CD4 (17-0041-82; 10 µg/ml), or CD8 (11-0081-82, 10 µg/ml), fixed in 2% paraformaldehyde, and permeabilized with 0.5% saponin before adding anti-IFN-γ (12-7311-82; 10 µg/ml) or control rat IgG1 (12-4301-82; 10 µg/ml). All antibodies were purchased from e-Bioscience. Samples were processed with a C6 Flow Cytometer instrument. Dead cells were excluded on the basis of forward and side light scatter. Data were analyzed with a CFlow Plus Flow Cytometer (BD Biosciences).
Cflow plus flow cytometer
Manufactured by BD
Sourced in United States
The CFlow Plus Flow Cytometer is a state-of-the-art instrument designed for high-performance cell analysis. It utilizes advanced flow cytometry technology to accurately measure and characterize various properties of cells in suspension, including size, granularity, and fluorescence. The CFlow Plus is capable of rapid multi-parameter data acquisition and analysis, enabling researchers to gain valuable insights into cell populations and their dynamics.
Automatically generated - may contain errors
Lab products found in correlation
Golgiplug, by BD (9 mentions)
Anti ifn γ, by Thermo Fisher Scientific (4 mentions)
Sars cov 2 s peptide pool, by Miltenyi Biotec (3 mentions)
Control rat igg1, by Thermo Fisher Scientific (3 mentions)
Anti tnf α, by Thermo Fisher Scientific (1 mentions)
Accuri c6 flow cytometer, by BD (1 mentions)
Collagenase type 4, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
C6 flow cytometer, by BD (1 mentions)
10 protocols using cflow plus flow cytometer
1
ZIKV-Specific T Cell Immune Response
2
Quantifying SARS-CoV-2 Specific T Cell Responses
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As described previously [28 (link)], splenocytes were incubated with SARS-CoV-2 S peptide pools (1 μg/mL, Miltenyi Biotec, Auburn, CA, USA) for 5 h in the presence of BD GolgiPlug (BD Biosciences, San Jose, CA, USA). Briefly, cells were stained with antibodies for CD4 or CD8, fixed in 2% paraformaldehyde, and permeabilized with 0.5% saponin before adding anti-IFN-γ, anti-TNF-α, or control rat IgG1 (e-Biosciences). Samples were processed with a C6 Flow Cytometer instrument. Dead cells were excluded based on forward and side light scatter. Data were analyzed with a CFlow Plus Flow Cytometer (BD Biosciences, San Jose, CA, USA).
3
ZIKV-Specific T Cell Responses
4
SARS-CoV-2 Spike Peptide-Induced T-cell Response
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Splenocytes or lung leukocytes were incubated with SARS-CoV-2 spike peptide pools (1 μg/ml, Miltenyi Biotec) for 6 h in the presence of GolgiPlug (BD Bioscience). Cells were stained with antibodies for CD3 (12-0031-81), CD4 (17-0041-82), or CD8 (11-0081-82) purchased from Thermo Fisher Scientific, fixed in 2% paraformaldehyde, and permeabilized with 0.5% saponin before adding anti-IFN-γ (12-7311-82, Thermo Fisher Scientific). Samples were processed with a C6 Flow Cytometer instrument. Dead cells were excluded based on forward and side light scatter (Supplementary Fig. 3 ). Data were analyzed with a CFlow Plus Flow Cytometer (BD Biosciences).
5
Zika Virus-Induced IFN-γ in T Cells
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Splenocytes (2.5 × 106) were incubated with 0.1 × 106 FFU live ZIKV-FSS13025 for 24 h. BD GolgiPlug (BD Bioscience) was added to block protein transport at the final 6 h of incubation. Cells were stained with antibodies for CD3, CD4, or CD8 fixed in 2% paraformaldehyde and permeabilized with 0.5% saponin before adding anti-IFN-γ, or control rat IgG1 (e-Biosciences). Samples were processed with a C6 Flow Cytometer instrument. Dead cells were excluded on the basis of forwarding and side light scatter. Data were analyzed with a CFlow Plus Flow Cytometer (BD Biosciences).
6
Zika Virus Stimulation of Splenocytes
7
SARS-CoV-2 Specific T-cell Assay
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Splenocytes were incubated with SARS-CoV-2 S peptide pools (1 μg/mL, Miltenyi Biotec) for 6 h in the presence of GolgiPlug (BD Bioscience). Cells were harvested and stained with antibodies for CD3, CD4, or CD8, fixed in 2% paraformaldehyde, and permeabilized with 0.5% saponin before adding anti-IFN-γ (Thermo Fisher Scientific) (24 (link), 31 (link)). Samples were acquired by a C6 Flow Cytometer instrument. Dead cells were excluded based on forward and side light scatter. Data were analyzed with a CFlow Plus Flow Cytometer (BD Biosciences).
8
SARS-CoV-2 T cell response assay
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Splenocytes or lung leukocytes were incubated with SARS-CoV-2 S peptide pools (1μg/ml, Miltenyi Biotec) for 24 h. BD GolgiPlug (BD Bioscience) was added to block protein transport at the final 6 h of incubation. Cells were stained with antibodies for CD3, CD4, or CD8, fixed in 2% paraformaldehyde, and permeabilized with 0.5% saponin before adding anti-IFN-γ, or control rat IgG1 (e-Biosciences). Samples were processed with a C6 Flow Cytometer instrument. Dead cells were excluded based on forward and side light scatter. Data were analyzed with a CFlow Plus Flow Cytometer (BD Biosciences).
9
ZIKV-Specific T Cell Immune Response
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Approximately 2.5 × 106 splenocytes were stimulated with 1 × 105 IFU of live ZIKV (strain FSS13025) for 24 h or 10 μg/ml E peptide (Sequence 294–302 in ZIKV polyprotein) [33 (link)] for 5 h. Live ZIKV was used as a stimulant for measuring both CD4+ and CD8+ T cell response [22 (link)]. The E peptide was used as stimulant for measuring CD8+ T cell response [33 (link)]. During the final 5 h of stimulation, BD GolgiPlug (BD Bioscience) was added to block protein transport. Cells were stained with antibodies against surface markers CD3 (APC-conjugated) and CD4 (FITC-conjugated) or CD8 (FITC-conjugated). Afterwards, cells were fixed in 2% paraformaldehyde and permeabilized with 0.5% saponin. Cells were then incubated with PE-conjugated anti-IFN-γ and PE-Cy7-conjugated anti-TNF-α antibodies or control PE-conjugated rat IgG1. Samples were processed with a BD Accuri™ C6 Flow Cytometer instrument. Dead cells were excluded on the basis of forward and side light scatter. Data were analyzed with a CFlow Plus Flow Cytometer (BD Biosciences).
10
Isolation and Flow Cytometric Analysis of Lung and Splenic Leukocytes
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Lung tissues were digested with 0.05% collagenase type IV (Thermo Fisher Scientific, Waltham, MA, USA) in RPMI 1640 Medium (Gibco, Billings, MT, USA) at 37 °C with 5% CO2 for 45 min, and a single-cell suspension was prepared by passing the lung homogenates through a 70 μm cell strainer followed by red blood cell lysis. Splenocytes or lung leukocytes were stained with antibodies for CD3 and TCRγδ (e-Biosciences, San Diego, CA, USA). After staining, the cells were fixed with 2% paraformaldehyde in PBS and examined using a C6 flow cytometer (BD Biosciences, San Jose, CA, USA). Live cells were enriched on the basis of forward and side light scatter. Data were analyzed with a CFlow Plus flow cytometer (BD Biosciences, San Jose, CA, USA).
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