To identify specific immune cell subsets in PBMCs following VitD3 treatment, cells were stained with fluorescently-conjugated monoclonal antibodies; CD4-BUV737, CD45RO-APC, CD161-FITC, CD194-V450, CD196-PE, CD14-BV605, CD19-APC-H7, CD56-BV421, CD282-AF647 (TLR2), CD284-BV786 (TLR4; all from BD Bioscience; San Diego, CA, USA), and anti-TLR7-PE (Gibco Life Technologies, Carlsbad, USA), Zombie Aqua™ Fixable Viability Kit (BioLegend, San Diego, USA). Compensation bead particles were used to account for spectral overlap (BD Bioscience, San Diego, CA, USA) and analyzed using the BD LSRII flow cytometer. Unstained PBMCs and fluorescence minus one (FMO) were used as controls and a minimum of 100,000 events were analyzed per sample gated on live, single cell lymphocyte gate based on FSC and SSC, where the expression of the cell surface molecules was evaluated using FlowJo, LLC v10.4.2 software. Refer to Supplementary Figures 1–4 for gating strategies.
Cd161 fitc
Manufactured by BD
Sourced in United States
CD161-FITC is a fluorescently-labeled monoclonal antibody that binds to the CD161 cell surface receptor. It is used for the identification and analysis of CD161-positive cells in flow cytometry applications.
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Lab products found in correlation
Cd4 buv737, by BD (1 mentions)
Cd196 pe, by BD (1 mentions)
Compensation bead particles, by BD (1 mentions)
Lsr 2 flow cytometer, by BD (1 mentions)
Cd19 apc h7, by BD (1 mentions)
Cd56 bv421, by BD (1 mentions)
Zombie aqua fixable viability kit, by BioLegend (1 mentions)
Cd14 bv605, by BD (1 mentions)
Cd45ro apc, by BD (1 mentions)
Cxcr6 apc, by BioLegend (1 mentions)
Facscanto 2, by BD (1 mentions)
Cd8 pe cy7, by BioLegend (1 mentions)
Cd45ro pe, by BD (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Cd69 fitc, by BD (1 mentions)
Cd25 pe, by BD (1 mentions)
Cd56 pe, by BD (1 mentions)
Cd16 pe, by BD (1 mentions)
Cd4 apc, by BD (1 mentions)
Cd3 pe, by BD (1 mentions)
4 protocols using cd161 fitc
1
Immunophenotyping of PBMC subsets following VitD3 treatment
2
Phenotyping immune cells by flow cytometry
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PBMCs or BACs from healthy individuals were stained with the monoclonal antibodies to the following human antigens: CD161-FITC, CD45RO-PE (BD Biosciences, San Jose, CA), CD4-APE-Cy7, CD8-PE-Cy7, CXCR6-APC (Biolegend San Diego, CA) or CD3-PE-TexRed (Invitrogen, Carlsbad, CA) and then by their respective IgG isotypes. Briefly, 2.5 x 105 PBMCs or BACs were incubated with antibody for 15 min at room temperature in the dark. Then, BACs and blood samples were washed twice with phosphate-buffered saline (PBS) containing 2% fetal bovine serum (wash solution). The supernatants were discarded and resuspended in PBS and 100,000 cells were acquired using a FACSCanto II flow cytometer (BD Biosciences). Data analysis was performed using FlowJo research software version 8.9 (Tree Star, Inc. Ashland, OR).
3
Phenotypic Analysis of CD8+ T Cells
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To measure the expression of different molecules on CD8+ cells, PBMCs were suspended in PBS containing 0.2% BSA and 0.1% sodium azide (PBS–BSA buffer). Cells were stained with the following monoclonal antibodies (mAbs): Phycoerythrin Cy5 (PECy5)‐CD8, Fluorescein isothiocyanate (FITC)‐TCRαβ, Phycoerythrin (PE)‐TCRγδ, PE‐NKG2D, FITC‐NKG2A, FITC‐CD69, PE‐CD25, PE‐CD56, FITC‐CD161, and PE‐CD16 (BD Biosciences Pharmingen, San Diego, CA), and then incubated for 20 min at 4°C. Data were collected using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA) and analyzed by FlowJo software (Tree Star, San Carlos, CA, USA). In each case, 20,000 events were acquired. Fluorochrome‐labeled isotype‐matched control mAbs were used to evaluate background staining.
4
Flow Cytometry Analysis of Immune Cells from Spleen and Aorta
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Rats were euthanized under anesthesia and single cell suspensions from spleens were obtained by disaggregating spleen tissue between frosted glass microscopy slides. Erythrocytes were lysed by suspending splenocytes pellets in a hypotonic solution (155 mM NH4Cl, 12 mM NaHCO3, 0.1 mM EDTA), nucleated cells were washed twice with phosphate buffered saline (PBS). Flow cytometry was performed as described earlier (Singh et al., 2017 (link)). Briefly, washed splenocytes were suspended in Fc blocking buffer (2% v/v fetal bovine serum and 1% v/v normal mouse serum in PBS). An aliquot of 106 cells was taken from each sample and mixed with FITC-CD161, PerCP-CD8a, APC-CD4, PE-CD3, and PE-Cy5-CD45RA antibodies (1 μl each antibody, all antibodies from BD Biosciences). After incubation with antibodies on ice for 30 min, cells were washed twice with PBS and resuspended in 400 μl PBS and flow cytometry was performed on a Beckton-Dickinson Aria flow cytometer. Thoracic aorta were minced with a razor blade and digested in 0.05% w/v Collagenase Type I and 0.05% w/v Collagenase Type II in HBSS. Cells were dissociated by trituration and filtered through a nylon membrane followed by two washes with ice cold PBS. Cells were then processed as described above for the spleen cells.
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