Lung cells were harvested from the layer of percoll gradients between 44% and 67% and stimulated with 4 μg/mL inactivated A/Cal H1N1 virus in the presence of Brefeldin A (20 μg/mL) for 5 hours at 37 °C as described (Lee et al., 2019 (link); Lee et al., 2018 (link)). In vitro cultured cells (lung and BAL cells) were stained with anti-CD3-PacificBlue (Clone 17A2, Biolegend, San Diego, CA), anti-CD4-PE/Cy5 (Clone RM405, BD Biosciences, San Jose, CA) antibodies, and then fixed and permeabilized using BD Cytofix/CytopermTM Plus Kit (BD Biosciences). After staining the cells with anti-IFN-γ-APC/Cy7 (Clone XMG1.2, BD), anti-TNF-α-PE/Cy7 (Clone MP6-XT22, Biolegend), and anti-Granzyme B-FITC (Clone NGZB, eBioscience) antibodies, live lymphocytes were first gated by forward versus side scatter strategic gating, followed by the gating of CD3+ T cells and then CD4 T cells secreting cytokines. The number of effector T cells in BAL and lung were expressed by reflecting the frequency gated out of the total cells from each mouse. Cells positive for intracellular cytokines were revealed through acquisition on a Becton-Dickinson LSR-II/Fortessa flow cytometer (BD, San Diego, CA) and analyzed by Flowjo software (Tree Star Inc., Ashland, OR).
Anti ifn γ apc cy7
Manufactured by BD
Anti-IFN-γ-APC-Cy7 is a fluorochrome-conjugated antibody that binds to the cytokine interferon-gamma (IFN-γ).
Automatically generated - may contain errors
Lab products found in correlation
Cytofix cytoperm plus kit, by BD (2 mentions)
Ionomycin, by Merck Group (2 mentions)
Anti tnf α pe cy7, by BioLegend (1 mentions)
Anti granzyme b fitc, by Thermo Fisher Scientific (1 mentions)
Anti cd3 pacific blue, by BioLegend (1 mentions)
Pe cy5 anti cd4, by BD (1 mentions)
Lsrii fortessa flow cytometer, by BD (1 mentions)
Anti il 4 pe, by BD (1 mentions)
Fix and perm cell permeabilization kit, by Thermo Fisher Scientific (1 mentions)
Monensin, by BD (1 mentions)
Anti il 10 pe, by BD (1 mentions)
Ant iil 4 pe cy7a, by BD (1 mentions)
Cytofix cytoperm fixation permeabilization solution kit, by BD (1 mentions)
Accucheck counting beads, by Thermo Fisher Scientific (1 mentions)
Phorbol myristate acetate (pma), by Enzo Life Sciences (1 mentions)
Cd16 cd32, by BioXCell (1 mentions)
Anti cd4 qdot605, by Thermo Fisher Scientific (1 mentions)
Live dead fixable aqua dead cell stain kit, by Thermo Fisher Scientific (1 mentions)
Brefeldin a, by BioLegend (1 mentions)
Anti cd3 pacific blue, by BD (1 mentions)
5 protocols using anti ifn γ apc cy7
1
Cytokine Profiling of Activated Lung T Cells
2
T Cell Cytokine Profiling via ICS
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For the ICS, T cells were stimulated with phorbol 12-myristate 13-acetate (50 ng/mL) and ionomycin (2 μg/mL; Sigma-Aldrich) for 5 hrs and with Monensin (1 μl/ml; BD Pharmingen) added for the final 4 hrs. Cells were pre-incubated with mouse IgG (20 μg) to block non-specific binding. Each sample was then labeled with anti-CD4-PerCP-Cy5.5 (1:200 μl) and anti-CD8-PE-Cy7 (1:200 μl) for 30 min. Cells were fixed by addition of 2% paraformaldehyde (100 μl) for 10 min followed by staining buffer (5% BSA and 0.1% sodium azide in PBS). ICS was performed with Fix and Perm cell permeabilization kit (Invitrogen) according to the manufacturer’s instructions. Cells were suspended in permeabilization buffer along with mouse IgG (50 μl). For cytokine staining, cells were incubated with either anti-IL-4-PE or anti-IFN-γ-APC-Cy7 (1:100) for 30 min (BD Biosciences, San Diego, CA). The cells were incubated for 30 min and washed with staining solution (5% BSA and 0.1% sodium azide in PBS). Flow cytometry was performed as described with appropriate gating on the CD4+ and CD8+ populations.
3
Spleen Cell Proliferation and Cytokine Profile
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proliferative response of CD4 and CD8 spleen cells after immunotherapy was assessed by flow cytometry as described15 ,40 (link). Results were expressed as proliferation index (PI) for CD4+ or CD8+ measured as the ratio: %CD4+CFSEdim or %CD8+CFSEdim in stimulated sample/%CD4+CFSEdim or %CD8+CFSEdim in non-stimulated sample and considered positive when the ratio was higher than 2.
The cytokine pattern was evaluated in spleen cells by determining intracellular cytokines using BD Cytofix/CytoPerm Fixation/Permeabilization solution kit (BD™ Biosciences) and staining with specific fluorochrome-conjugated mAbs, anti-IFNγ-APCCy7, ant-iIL-4-PE-Cy7A and anti-IL-10-PE (BD Pharmingen). Regulatory cells were defined as CD3+CD4+CD25+FoxP3+ using anti-CD25 and FoxP3 (BD Pharmingen). Results are expressed as percentages.
The cytokine pattern was evaluated in spleen cells by determining intracellular cytokines using BD Cytofix/CytoPerm Fixation/Permeabilization solution kit (BD™ Biosciences) and staining with specific fluorochrome-conjugated mAbs, anti-IFNγ-APCCy7, ant-iIL-4-PE-Cy7A and anti-IL-10-PE (BD Pharmingen). Regulatory cells were defined as CD3+CD4+CD25+FoxP3+ using anti-CD25 and FoxP3 (BD Pharmingen). Results are expressed as percentages.
4
Multiparametric Flow Cytometry Profiling
5
Multiparameter Flow Cytometry of Splenocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Splenocytes (2 × 106 cells/well) isolated at 8 weeks after immunization were plated in 96-well plates and stimulated with 10 μg/mL PPD for 14 h in the presence of 1 μg/mL anti-CD28 (BD Biosciences) and subsequently incubated for an additional 5 h at 37°C following the addition of 0.5 μL/mL monensin/GolgiStop (BD Biosciences). Following overnight incubation at 4°C, the cells were washed in FACS buffer (PBS containing 0.1% sodium azide and 1% FBS) and subsequently stained for 30 min at 4°C for surface markers with mAbs as indicated using anti-CD3-Pacific Blue, anti-CD8-FITC, and anti-CD44-V500 (all from BD Biosciences). Cells were then washed in FACS buffer, fixed, permeabilized using the Cytofix/Cytoperm kit (BD Biosciences) according to the manufacturer's instructions and stained intracellularly for 30 min at 4°C using anti-IFN-γ-APC-Cy7, anti-TNF-α-Percp-Cy5.5, and anti-IL-2-APC mAbs (all from BD Biosciences). Cells were subsequently washed, resuspended in FACS buffer, and then analyzed by multiparameter flow cytometry using a BD FACSAria flow cytometer (BD Biosciences). For each sample, at least 300,000 events were collected and responses were analyzed using FlowJo software (Tree Star, USA).
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!