Surface and intracellular staining for flow cytometry analysis were performed as described previously [19 (link),20 ]. The antibodies used for surface and intracellular staining are listed and their Research Resource Identifiers (RRID) tags are provided in Table S3 . For surface staining, cells were incubated with relevant fluorochrome-labelled antibodies for 20 min at 4 °C in the dark. For intracellular cytokine staining (ICS), cells were fixed and permeabilized using the Intracellular Fixation & Permeabilization Buffer Set (Invitrogen, USA) and stained with FITC-anti-IFN-γ, PE-anti-IL-2, or APC-anti-TNF-α (eBioscience, San Diego, USA). Freshly isolated cells were used for all assays, and approximately 20 000–40 000 T cells were acquired for each sample using a BD FACS Canto II flow cytometer. Data analysis was performed using Flow Jo software V10.0.7 (Tree Star, Ashland, OR, USA). Cell debris and dead cells were excluded from the analysis based on scatter signals and Fixable Viability Dye eFluor 506 (Thermo Fisher Scientific).
Pe anti il 2
Manufactured by Thermo Fisher Scientific
Sourced in United States
PE-anti-IL-2 is a fluorescently-labeled monoclonal antibody that binds to interleukin-2 (IL-2), a cytokine involved in immune cell activation and proliferation. This product is designed for use in flow cytometry applications to detect and quantify IL-2 expressing cells.
Automatically generated - may contain errors
Lab products found in correlation
Anti ifn γ fitc, by Thermo Fisher Scientific (4 mentions)
Intracellular fixation permeabilization buffer set, by Thermo Fisher Scientific (3 mentions)
Celltrace violet, by Thermo Fisher Scientific (3 mentions)
Facscanto 2 flow cytometer, by BD (2 mentions)
Anti tnf α apc, by Thermo Fisher Scientific (2 mentions)
Live dead fixable aqua dead cell stain kit, by Thermo Fisher Scientific (2 mentions)
Anti cd62l pe cy7, by Thermo Fisher Scientific (2 mentions)
Apc anti tnfα, by Thermo Fisher Scientific (1 mentions)
Fixable viability dye efluor 506, by Thermo Fisher Scientific (1 mentions)
Anti ifn γ pe, by Thermo Fisher Scientific (1 mentions)
Anti cd11b bv510, by BD (1 mentions)
Anti cd3 bv510, by BD (1 mentions)
Anti cd16 32, by BD (1 mentions)
Pe anti f4 80, by BD (1 mentions)
Anti cd11c apc, by Thermo Fisher Scientific (1 mentions)
Anti il 4 apc, by Thermo Fisher Scientific (1 mentions)
Lsrfortessa, by BD (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
Facscalibur flow cytometer system, by BD (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
8 protocols using pe anti il 2
1
Multiparametric Flow Cytometry of T Cells
2
Multiparametric Immune Cell Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Splenocytes and lung cells were incubated with anti-CD16/32 (BD Bioscience, USA) for 30min at 4°C in PBS supplemented with 2% FBS (FACS buffer) to block non-specific binding with Fc receptors. Cells were surface-stained with BV650-anti-CD8 (BD-563234, USA), BV711-anti-CD4 (BD-563050, USA), BV510anti-CD3 (BD-563024, USA), PECY7-anti-CD25 (eBioscience-25-0251-82, USA), PE-anti-F4/80 (BD-565410, USA), BV510-anti-CD11b (BD-562950, USA), APC-anti-CD11c (eBioscience-17-0114-81, USA) for 20 min on ice and washed twice with PBS. For intracellular staining, cells were fixed and permeablized using BD CytoFix/CytoPerm Kit (BD Bioscience, USA) and stained with APC-anti-IL-4 (eBioscience-17-7041-82, USA), PE-anti-IFN-γ (eBioscience-12-7311-82, USA), PE-anti-IL-2 (eBioscience-12-7021-82, USA), BV421-anti-IL-17 (BD-563354, USA), PECY7-anti-TNF-ɑ (BD-557644, USA), Pacific blue-anti-Foxp3 (eBioscience-48-5773-82, USA) for 20min. Cells were washed with 1×BD Perm/Wash buffer and were finally resuspended in 4% paraformaldehyde (PFA) for flow cytometry analysis. Flow cytometry was performed using BD LSR Fortessa (BD Bioscience, USA) and raw data was analyzed using FlowJo software.
3
Comprehensive Flow Cytometry Immunophenotyping
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Surface and intracellular staining for flow cytometry analysis were performed as described previously (Liu et al., 2013 (link); Wang et al., 2018 (link)). For surface staining, cells were incubated with relevant fluorochrome-labeled antibodies (eFluor 780-anti-CD3, PE-Cy7-anti-CD8, and PerCP-Cy5.5-anti-CD4) for 30 min at 4 °C in the dark. For intracellular cytokine staining, cells were fixed and permeabilized using the Intracellular Fixation & Permeabilization Buffer Set (Invitrogen, USA) and subsequently stained with FITC-anti–IFN–γ, PE-anti-IL-2 and APC-anti-TNF-α (Invitrogen, USA). Approximately 100,000 PBMCs were acquired for each sample using a BD FACS Canto II flow cytometer. Data analysis was performed using the FlowJo software V10.0.7 (Tree Star, Ashland, OR, USA). Cell debris and dead cells were excluded from the analysis based on scatter signals and Fixable Viability Dye eFluor 506.
4
Intracellular Cytokine Staining of Alloactivated CD4+ T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To determine cytokine production by alloactivated responding CD4+ T cells on a single cell level we stained the cells intracellularly, using FITC-anti-IFN-γ, PE-anti-IL-10, PE-anti-IL-4 and PE-anti-IL-2 Abs (all from Invitrogen, Carlsbad, CA). For gating purposes we used Cy5-PE-anti-CD4 Ab (Biolegend, CA, USA). Briefly, 1 × 105 DCs (either immature or mature, from single and mixed DC cultures) were co-cultured with 1 × 106 nal¨ve CD4+CD45RA+ T cells in 24-well tissue culture plates in complete medium (X-VIVO15 supplemented with 1% human AB serum). After 7 days, CD4+ T cells were collected and restimulated with 50 ng/ml phorbol myristate acetate (PMA) and 500 ng/ml ionomycin (both from Sigma). After 4 hours 10 µg/ml of Brefeldin A (Biolegend) were added for an additional 4 hours. Afterwards the cells were fixed with 4% PHA for 1 hour and then permeabilized using 0.1% Triton X-100 for 10 minutes. Subsequently the cells were washed twice with DPBS. To prevent non-specific staining, T cells were incubated for 45 min in DPBS containing 3% bovine serum albumin (BSA) and then stained with FITC-anti-IFN-γ, PE-anti-IL-10, PE-anti-IL-4 and PE-anti-IL-2 Abs for 45 min in the dark. Before they were analyzed with the FACSCalibur flow cytometer system (Becton Dickinson, Inc.), the cells were washed twice with DPBS.
5
Flow Cytometry Analysis of Immune Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Surface and intracellular staining for flow cytometry analysis were performed as described previously (13 (link), 14 (link)). For surface staining, cells were incubated with relevant fluorochrome-labeled antibodies (eFluor 780-anti-CD3, PE-Cy7-anti-CD8, and PerCP-Cy5.5-anti-CD4) for 30 min at 4°C in the dark. For intracellular cytokine staining, cells were fixed and permeabilized using the Intracellular Fixation & Permeabilization Buffer Set (Invitrogen, USA) and subsequently stained with FITC-anti-IFN-γ, PE-anti-IL-2 and APC-anti-TNF-α (Invitrogen, USA). Approximately 100,000 PBMCs were acquired for each sample using a BD FACS Canto II flow cytometer. Data analysis was performed using the FlowJo software V10.0.7 (Tree Star, Ashland, OR, USA). Cell debris and dead cells were excluded from the analysis based on scatter signals and Fixable Viability Dye eFluor 506.
6
Profiling Antigen-Specific T-cell Responses
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Splenocytes were prepared as described above, plated in 96-well round-bottom plates, and stimulated using peptide pools for EBOV GP, SUDV GP, or MARV GP (as described above) at a final concentration of 5 µg/mL or media only. Stimulation and staining was then performed as described previously [33 (link)] except that the following antibodies were used: anti-CD4-Qdot605, anti-CD127-APCef780 (Invitrogen), anti-CD62L-PeCy7, and anti-CD8-PerCP/Cy5.5 antibodies (eBioscience), as well as LIVE/DEAD® Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific), anti-TNF-Alexa488, anti-IL-2-PE, and anti-IFN-γ-e450 antibodies (eBioscience). Antigen-specific cells were identified by gating based on doublet negative, size, live cells, and either CD4+ or CD8+ surface expression. Background responses in unstimulated control samples were subtracted from responses of peptide stimulated T cells.
7
LASV-specific T-cell Responses Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Splenocytes were isolated for analysis via IFN-γ ELISpot, surface and intracellular cytokine staining (ICS) and flow cytometry as previously described69 (link)–71 (link). Splenocytes were restimulated with 2 μg/mL of the appropriate antigenic peptide pool (comprised of individual peptides that were 20 amino acids in length with 10 amino acids of overlap) spanning the full-length LASV GPC sequence (Mimotopes). Reference sequences for peptide synthesis are as follows: NP_694870.1 (Josiah), AIT17836.1 (Pinneo), AAF86703.1 (803213), CAA36645.1 (GA391). Cells were restimulated for 18–20 h or 6 h (at 37 °C and 5% CO2) for IFN-γ ELISpot and ICS, respectively. Surface staining and ICS were carried out using the following antibodies: LIVE/DEAD® Fixable Aqua Dead Cell Stain Kit (Thermo Fischer Scientific); anti-CD8a-PerCP/Cy5.5, anti-CD62L-PeCy7, anti-IFN-γ-eFluor 450, anti-TNF-α-Alexa Fluor 488, anti-IL-2-PE (eBioscience); anti-CD4-Brilliant Violet 650, anti-CD44-Alexa Fluor 700 (BioLegend); and anti-CD127-eFluor 660 (Invitrogen). Antigen-specific T-cell responses were quantified by subtracting the response (SFU for IFN-γ ELISpot and the percentage of cytokine-positive cells for ICS) measured without stimulation from that observed after restimulation.
8
Barcoded Cell Calcium Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To decrease experimental variation, cell lines were barcoded by differential labeling with fourfold dilutions of CellTrace Far Red or CellTrace Violet (ThermoFisher), and mixed together prior to stimulation as described [42] . For calcium assays, mixed, CellTrace Far Red-barcoded cells were loaded with the fluorescent calcium indicator dye, Indo1-AM (eBioscience), and then washed twice and resuspended in calcium buffer, consisting of 25 mM Hepes (pH 7.4), 1 mM CaCl2, 125 mM NaCl, 5 mM KCl, 1 mM Na2HPO4, 0.5mM MgCl2, 1 g/l glucose, 2 mM L-glutamine and 1 mg/ml high-purity bovine serum albumin (Sigma A4378). Intracellular calcium was measured by FACS at 37°C, with C305 or C305+CD28.2 stimulant added at the 60s time point [38] . CellTrace Violet-barcoded cells were stimulated as indicated in the figure legends, and stained with anti-CD69, or were fixed, permeabilized and stained with anti-pJNK, or with anti-IL-2-PE (eBioscience). Results were analyzed using FlowJo, while gating on a defined GFP window within each barcoded population.
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!