Spleen cells from mice were collected by splenic grinding, each step with 5 mL RPMI‐1640 (Gibco). Cells were subsequently washed twice and then resuspended in RPMI‐1640. For peptide stimulation, cells were incubated overnight with individual peptides or a mixture of the 3 peptides in the presence of Brefeldin A. Activation cocktail (Biolegend) was used as a positive control. Background stimulation was assessed by stimulation with PBS control. Antigen-specific CD8 + T cells were analyzed by staining for CD3 (clone 17A2), CD8 (clone 53–6.7), and intracellular IFN-γ (clone XMG1.2) using a flow cytometer in the Johns Hopkins flow cytometry core. Data were acquired and analyzed using BD FACSDiva™ software.
Activation cocktail
Manufactured by BioLegend
Sourced in United States
Activation cocktail is a laboratory reagent used to activate immune cells, such as T cells or B cells, in cell culture experiments. It contains a combination of stimulatory molecules that trigger cellular activation and proliferation. The specific composition and intended use of the activation cocktail may vary depending on the research application.
Automatically generated - may contain errors
Lab products found in correlation
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Facsdiva software, by BD (1 mentions)
Pe conjugated anti cd25, by BioLegend (1 mentions)
Pe cy7 conjugated anti ifn γ, by BioLegend (1 mentions)
Alexa fluor 647 conjugated anti foxp3, by BioLegend (1 mentions)
Apc conjugated anti il 17a, by BioLegend (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Facscanto 2, by BD (1 mentions)
Monensin, by BioLegend (1 mentions)
Fixation permeabilization solution kit, by BD (1 mentions)
Anti mouse cd16 32, by BioLegend (1 mentions)
Fitc conjugated anti mouse cd4 antibody, by BioLegend (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
Phytohemagglutinin m, by Merck Group (1 mentions)
Anti human cd28, by Miltenyi Biotec (1 mentions)
Sars cov 2 peptide pools, by Miltenyi Biotec (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Brefeldin a, by BioLegend (1 mentions)
5 protocols using activation cocktail
1
Murine Spleen T Cell Stimulation
2
Quantifying T-cell Subsets in Mice
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Spleens and blood from the mice were harvested, and a single cell suspension was prepared. For quantification of the number of Th1/Th2/Th17 cells, cells were stimulated with PMA (50 ng/ml, Sigma), activation cocktail (750 ng/ml, BioLegend), and monensin (2 μmol/L, BioLegend). Cells were thereafter stained with FITC-conjugated anti-CD4 antibodies (BioLegend), permeabilized with permeabilization solution, and then stained with PE/Cy7-conjugated anti-IFN-γ, PE-conjugated anti-IL-4 and APC-conjugated anti-IL-17A (BioLegend). For detection of Treg cells, the cells were stained with PE-conjugated anti-CD25 and Alexa Fluor 647-conjugated anti-Foxp3 (BioLegend). Fluorescence was examined with FACS Canto II (BD Biosciences). All flow cytometry data presented herein were gated by the use of CD4+, while IFN-γ+ cells represented Th1 cells, IL-4+ cells represented Th2 cells, IL-17A+ cells represented Th17 cells, and CD25+Foxp3+ cells represented Treg cells. Data were analyzed with the FlowJo software.
3
Isolation and Characterization of IL-17A+ T cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Following the perfusion, isolated brains were dissociated and chopped into small pieces, and then they were filtered through a 70 μm cell strainer and centrifuged at 1300 rpm for 5 min. The cell suspension was mixed with 30% Percoll/HBSS medium, overlaid on 70% Percoll/HBSS medium and then centrifuged at 2000 rpm for 30 min without using the brake. After the cells between the 30/70% Percoll gradients were recovered, they were washed once. The cells in media were stimulated with activation Cocktail (BioLegend, San Diego, CA, USA) for 4 h at the recommended concentrations. The cells were blocked with a purified anti-mouse CD16/32 (BioLegend) antibody and stained extracellularly with a PerCP/Cyanine5.5-conjugated anti-mouse CD45 antibody (BioLegend) and FITC-conjugated anti-mouse CD4 antibody (BioLegend). To intracellularly stain the cells, they were fixed and permeabilized using a Fixation/Permeabilization Solution Kit (BD Bioscience, San Jose, CA, USA) and stained with an Alexa Fluor 647-conjugated anti-mouse IL-17A antibody (BioLegend). Data were acquired using an LSRII flow cytometer (BD Biosciences) and analyzed with FlowJo software version 8.7 (FlowJo, LCC, Ashland, OR, USA).
4
Evaluating LAG-3 Targeting Probes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immune cells from C57BL/6 mice and human peripheral blood mononuclear cells (hPBMCs; catalog no. HPA01091; Hope Biotechnology) were used. Immune stimulation with an activation cocktail (catalog no. 423301; BioLegend) on C57BL/6 mouse-derived immune cells and phytohemagglutinin-M (catalog no. 11082132001; Sigma) on hPBMCs was performed for flow cytometry analysis of LAG-3 and CD8. Cell uptake of [ 68 Ga]Ga-NOTA-C25 or [ 68 Ga]Ga-NOTA-scrambled C25 (0.037 MBq/mL; 6.788 3 10 29 mM) was performed by blocking assays with nonradiolabeled C25 (9.613 3 10 26 mM) and anti-LAG-3 antibody (aLAG-3; 1.333 3 10 27 mM) to test probe specificity. Detailed protocols are provided in the supplemental materials.
5
SARS-CoV-2 T Cell Stimulation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
In vitro T cell stimulation assays were carried out with spike (S), membrane (M), and nuclear (N) structural proteins. Briefly, viable cell numbers were determined in the thawed PBMCs by staining with crystal violet and counting with a hemocytometer. For the assays, 106 cells were resuspended in 100 μL RPMI 1640 medium (Gibco) supplemented with 10% heat-inactivated FBS and 1% penicillin/streptomycin. The SARS-CoV-2 peptide pools (Miltenyi Biotec, Germany) were prepared according to the manufacturer’s recommendations. Next, 1 μg of peptide/mL (0.6 nmol) separately or in a mixture was introduced to the T cells. Along with the peptide pools, 0.1 μg/mL purified anti-human CD28 (Miltenyi Biotec, Clone: REA612) and 0.1 μg/mL purified anti-human CD49d (Miltenyi Biotec, Clone: MZ18-24A9) as coactivators of T cells were also added to the wells for the entire stimulation period. The T cells and peptide mixtures were incubated at 37°C in 5% CO2 for 16 hours. Brefeldin A (Biolegend, San Diego, CA) at a concentration of 0.1 μg/mL was added to the culture medium in the last 4 hours to enhance intracellular cytokine staining signals. The negative control was 10% DMSO and the positive control was an activation cocktail (Biolegend) containing 8.1 nM phorbol-12-myristate (PMA) and 1.3 mM ionomycin.
+ Expand
In vitro T cell stimulation assays were carried out with spike (S), membrane (M), and nuclear (N) structural proteins. Briefly, viable cell numbers were determined in the thawed PBMCs by staining with crystal violet and counting with a hemocytometer. For the assays, 106 cells were resuspended in 100 μL RPMI 1640 medium (Gibco) supplemented with 10% heat-inactivated FBS and 1% penicillin/streptomycin. The SARS-CoV-2 peptide pools (Miltenyi Biotec, Germany) were prepared according to the manufacturer’s recommendations. Next, 1 μg of peptide/mL (0.6 nmol) separately or in a mixture was introduced to the T cells. Along with the peptide pools, 0.1 μg/mL purified anti-human CD28 (Miltenyi Biotec, Clone: REA612) and 0.1 μg/mL purified anti-human CD49d (Miltenyi Biotec, Clone: MZ18-24A9) as coactivators of T cells were also added to the wells for the entire stimulation period. The T cells and peptide mixtures were incubated at 37°C in 5% CO2 for 16 hours. Brefeldin A (Biolegend, San Diego, CA) at a concentration of 0.1 μg/mL was added to the culture medium in the last 4 hours to enhance intracellular cytokine staining signals. The negative control was 10% DMSO and the positive control was an activation cocktail (Biolegend) containing 8.1 nM phorbol-12-myristate (PMA) and 1.3 mM ionomycin.
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!