Levels of cytokines and transcription factors were assessed by intracellular staining using anti-IL-17-FITC, anti-Foxp3-FITC, and anti-Foxp3-PE antibodies (all from eBioscience, San Diego, CA, USA). Cells were stimulated with phorbol myristate acetate and ionomycin with the addition of GolgiStop for 4 h. Cultured cells were surface labeled for 30 min and permeabilized with Cytofix/Cytoperm solution (BD Pharmingen, Heidelberg, Germany). Cells were intracellularly stained with fluorescent antibodies and subjected to flow cytometry (FACSCalibur; BD Biosciences, Franklin Lakes, NJ, USA). Events were collected and analyzed using FlowJo software (Tree Star, Ashland, OR, USA).
Anti il 17 fitc
Manufactured by Thermo Fisher Scientific
Sourced in United States
Anti-IL-17-FITC is a fluorescently labeled antibody that binds to the cytokine interleukin-17. It is designed for use in flow cytometry applications to detect and analyze cells expressing IL-17.
Automatically generated - may contain errors
Lab products found in correlation
Facscalibur, by BD (5 mentions)
Anti foxp3 fitc, by Thermo Fisher Scientific (4 mentions)
Cytofix cytoperm solution, by BD (4 mentions)
Anti foxp3 pe, by Thermo Fisher Scientific (4 mentions)
Alpha minimal essential medium, by Thermo Fisher Scientific (2 mentions)
Anti cd3 cd28 beads, by Thermo Fisher Scientific (1 mentions)
Anti il 5 pe, by BioLegend (1 mentions)
Brefeldin a, by BioLegend (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Transcription factor buffer set, by BD (1 mentions)
Anti ifn γ fitc, by BioLegend (1 mentions)
Anti cd45.2 pe clone 104, by Thermo Fisher Scientific (1 mentions)
Anti gr 1 apc, by BioLegend (1 mentions)
Anti cd11b fitc, by BioLegend (1 mentions)
Facscanto flow cytometer, by BD (1 mentions)
6 protocols using anti il 17 fitc
1
Intracellular Cytokine and Transcription Factor Profiling
2
Intracellular Cytokine Profiling of Memory CD4+ T Cells
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Intracellular staining of FoxP3, IFNγ, IL-5, IL-17, and IL-10 was performed in CD4+ T cells from HD purified by negative selection using CD4+CD45RO+ microbeads kit following the protocol of manufacturer (Miltenyi Biotec). Memory T CD4+ cells were sorted in FACSAria III obtaining more than 98% purity. Cells were recovered overnight with RPMI complete media and the next day 1 × 106 memory CD4+ T cells were challenged with ANDV-GP VLP in presence of 2.5 μL of anti-CD3/CD28 beads (ThermoFisher Scientific), also, an anti-CD3/CD28 condition was included and without stimuli was used as control (mock). After 3 days, cells were stimulated with 50 ng/ml phorbol 12-myristate 13-acetate (PMA) and 1 μg/ml ionomycin (Sigma Aldrich) in presence of 1 μL brefeldin A (Biolegend) for 5 h. Later, cells were stained with fixable viability stain AF780 and fixed/permeabilized with transcription factor buffer set following manufacturer instructions (BD Biosciences). Intracellular staining was performed using the following antibodies: anti-IFNγ-FITC (Biolegend), anti-IL-5-PE (BioLegend), anti-FoxP3-eFluor450 or anti-IL-10-PE, and anti-IL-17-FITC (eBioscience).
3
Cytokine and Transcription Factor Analysis
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Expression levels of cytokines and transcription factors were assessed by intracellular staining using the following antibodies. For intracellular staining: anti-IL-17-FITC, anti-Foxp3-FITC, and anti-Foxp3-PE (all from eBioscience). Cells were stimulated with PMA and ionomycin with the addition of GolgiStop for 4 h. Cultured cells were surface labeled for 30 min and permeabilized with Cytofix/Cytoperm solution (BD Pharmingen, Heidelberg, Germany). Cells were intracellularly stained with fluorescent antibodies before flow cytometry (FACSCalibur; BD Biosciences, Franklin Lakes, NJ). Events were collected and analyzed with FlowJo software (Tree Star, Ashland, OR).
4
Intravascular Leukocyte Discrimination Protocol
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Intravascular staining for the discrimination of vascular and lung tissue leukocytes was conducted as previously described [26 (link)]. Anti-CD45.2 PE (clone 104, catalog number 12-0454-81; eBioscience, San Diego, California) was diluted to 25 mg/mL in sterile PBS; 100 μL of the solution was intravenously injected via the mouse tail 3 minutes before sacrificing the mice. The stained CD45.2+ cells are blood-borne in the mouse lungs and can be discriminated from cells localized in the lung tissue. Cellular staining and flow cytometry analyses for T helper (Th) cells and neutrophils were conducted as previously described [27 (link)]. In brief, cells were stained for surface markers with anti-CD4 fluorescein isothiocyanate (FITC; clone: GK1.5; eBioscience) for Th cells, and with anti-CD11b FITC (clone: M1/70; Biolegend, San Diego, California) and anti-Gr-1 APC (clone: RB6-8C5; Biolegend) for neutrophils. For intracellular staining, fixed cells were permeabilized and stained with anti–IL-17 FITC (catalog number 11-7177-81; eBioscience) and anti–interferon gamma (IFN-γ) APC (clone: XMG1.2; eBioscience) for 60 minutes in the dark at 4°C. Cells were washed with 1 mL of permeabilization buffer and resuspended in staining buffer for analysis. Samples were analyzed using a FACSCalibur or FACSCanto flow cytometer (BD Biosciences) and the FlowJo software (Tree Star, Ashland, Oregon).
5
Quantifying Th17 and Treg Cells by Flow Cytometry
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Levels of cytokines and transcription factors were assessed by intracellular staining using anti-IL-17-FITC, anti-Foxp3-FITC, and anti-Foxp3-PE antibodies (all from eBioscience, San Diego, CA, USA). Cells were stimulated with phorbol myristate acetate and ionomycin with the addition of GolgiStop for 4 h. Cultured cells were surface labeled for 30 min and permeabilized with Cytofix/Cytoperm solution (BD Pharmingen, Heidelberg, Germany). Cells were intracellularly stained with fluorescent antibodies and subjected to flow cytometry (FACSCalibur; BD Biosciences, Franklin Lakes, NJ, USA). Events were collected and analyzed using FlowJo software (Tree Star, Ashland, OR, USA).
In vitro osteoclastogenesis and tartrate-resistant acid phosphatase staining Bone marrow cells from mouse femurs were cultured in alpha-minimal essential medium (Invitrogen, Carlsbad, CA, USA) containing antibiotics and 10% heat-inactivated fetal bovine serum to separate oating and adherent cells. Nonadherent cells were removed by washing with media, and preosteoclasts were cultured in the presence of 10 ng/mL macrophage colony-stimulating factor, 100 ng/mL receptor activator of nuclear factor kappa-Β (RANK) ligand (RANKL) (PeptoTech, London, UK), and L. sakei for 4 days to generate osteoclasts. The medium was changed every 2 days. Osteoclasts were generated after 8-10 days.
In vitro osteoclastogenesis and tartrate-resistant acid phosphatase staining Bone marrow cells from mouse femurs were cultured in alpha-minimal essential medium (Invitrogen, Carlsbad, CA, USA) containing antibiotics and 10% heat-inactivated fetal bovine serum to separate oating and adherent cells. Nonadherent cells were removed by washing with media, and preosteoclasts were cultured in the presence of 10 ng/mL macrophage colony-stimulating factor, 100 ng/mL receptor activator of nuclear factor kappa-Β (RANK) ligand (RANKL) (PeptoTech, London, UK), and L. sakei for 4 days to generate osteoclasts. The medium was changed every 2 days. Osteoclasts were generated after 8-10 days.
6
Quantifying Th17 and Treg Cells by Flow Cytometry
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Levels of cytokines and transcription factors were assessed by intracellular staining using anti-IL-17-FITC, anti-Foxp3-FITC, and anti-Foxp3-PE antibodies (all from eBioscience, San Diego, CA, USA). Cells were stimulated with phorbol myristate acetate and ionomycin with the addition of GolgiStop for 4 h. Cultured cells were surface labeled for 30 min and permeabilized with Cytofix/Cytoperm solution (BD Pharmingen, Heidelberg, Germany). Cells were intracellularly stained with fluorescent antibodies and subjected to flow cytometry (FACSCalibur; BD Biosciences, Franklin Lakes, NJ, USA). Events were collected and analyzed using FlowJo software (Tree Star, Ashland, OR, USA).
In vitro osteoclastogenesis and tartrate-resistant acid phosphatase staining Bone marrow cells from mouse femurs were cultured in alpha-minimal essential medium (Invitrogen, Carlsbad, CA, USA) containing antibiotics and 10% heat-inactivated fetal bovine serum to separate oating and adherent cells. Nonadherent cells were removed by washing with media, and preosteoclasts were cultured in the presence of 10 ng/mL macrophage colony-stimulating factor, 100 ng/mL receptor activator of nuclear factor kappa-Β (RANK) ligand (RANKL) (PeptoTech, London, UK), and L. sakei for 4 days to generate osteoclasts. The medium was changed every 2 days. Osteoclasts were generated after 8-10 days.
In vitro osteoclastogenesis and tartrate-resistant acid phosphatase staining Bone marrow cells from mouse femurs were cultured in alpha-minimal essential medium (Invitrogen, Carlsbad, CA, USA) containing antibiotics and 10% heat-inactivated fetal bovine serum to separate oating and adherent cells. Nonadherent cells were removed by washing with media, and preosteoclasts were cultured in the presence of 10 ng/mL macrophage colony-stimulating factor, 100 ng/mL receptor activator of nuclear factor kappa-Β (RANK) ligand (RANKL) (PeptoTech, London, UK), and L. sakei for 4 days to generate osteoclasts. The medium was changed every 2 days. Osteoclasts were generated after 8-10 days.
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