Isolated PBMCs where phenotyped by staining with the following cell surface mAbs: CD3-APC efluor780 (SK7, eBioscience, San Diego, CA), CD4-Qdot655 (S3.5, Invitrogen, Carlsbad, CA), CD8-V500 (RPA-T8, BD Biosciences, Franklin Lake, NJ), and CD14-Pecy7 (M-5E2, BD Biosciences). LIVE/DEAD- Fixable Aqua Stain (Invitrogen, Carlsbad, CA) was used to exclude dead cells from analysis. Intracellular staining for antibodies recognizing CysB-FITC (Assaypro, St. Charles, MO) and CatB-PE (Cell Signaling, Danvers, MA) was performed after permeabilizing and fixing cells with the BD Cytofix/Cytoperm kit according to the manufacturer’s protocols. Supplemental Digital Content Figures 9 and 10 illustrate the gating strategy used. All antibody-stained cells were fixed in 1% formaldehyde (Sigma, St Louis, MO) prior to sample acquisition on a LSR II flow cytometer (BD Biosciences). We ran at least 100,000 gated lymphocytes/monocytes for each stained specimen. Gates for flow cytometric acquisition and analyses were based on isotype controls and single stain compensation controls. Antibody expression was measured via mean fluorescent intensity (MFI). Data were analyzed using FlowJo Version 9.9 software for Mac (TreeStar, San Carlos, CA). Cell viability was >70% across all participant groups.
Cd3 apc efluor780 sk7
Manufactured by Thermo Fisher Scientific
The CD3-APC efluor780 (SK7) is a fluorescently-labeled antibody product manufactured by Thermo Fisher Scientific. It is designed for use in flow cytometry applications to identify and analyze T cells. The product provides a specific labeling of the CD3 cell surface marker, which is expressed on T cells.
Automatically generated - may contain errors
Lab products found in correlation
Cytofix cytoperm kit, by BD (3 mentions)
Live dead fixable aqua stain, by Thermo Fisher Scientific (2 mentions)
Cd8 v500 rpa t8, by BD (2 mentions)
Cd14 pecy7 m 5e2, by BD (1 mentions)
Formaldehyde, by Merck Group (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
Granzyme b bv421, by BD (1 mentions)
Symphony flow cytometer, by BD (1 mentions)
Ifn γ pe cy7 b27, by BD (1 mentions)
Tnf α apc, by BD (1 mentions)
Cd69 fitc fn50, by BD (1 mentions)
Cd4 bv786 sk3, by BioLegend (1 mentions)
Facscanto 2, by BD (1 mentions)
3 protocols using cd3 apc efluor780 sk7
1
PBMCs Phenotyping and Intracellular Staining
2
Comprehensive T Cell Immunophenotyping
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LIVE/DEAD Fixable Aqua Stain (Invitrogen), excited by violet 405 nm laser and detected by 512 nm emission channel, was used to determine the percentage of dead cells. The T cell phenotype was determined by staining with mAbs: CD3-APC efluor780 (SK7, eBiosciences), CD4-BV786 (SK3, Biolegend), CD8-V500 (RPA-T8, BD Biosciences), and CD28-BV711 (CD28.2, BD Biosciences). Activation status was determined using mAbs: PD-1-PE-eFlour610 (J105, eBioscience), CD38-BUV737 (HB7, BD Biosciences), CD69-FITC (FN50, BD Biosciences), and CD25-BUV395 (2A3, BD Biosciences). Cytolytic profile was determined using mAbs: TNF-α-APC (6401.1111, BD Biosciences), Granzyme B-BV421 (GB11; BD Biosciences), and IFN-γ-PE-cy7 (B27, BD Biosciences). Monoclonal Ab CD14 BUV805 (M5E2, BD Biosciences, Franklin Lake, NJ) was used to exclude monocytes from analysis. Intracellular staining (ICS) was performed using the BD Cytofix/Cytoperm kit according to the manufacturer’s protocols. All antibody-stained cells were fixed in 1% formaldehyde (Sigma) prior to sample acquisition on a Symphony flow cytometer (BD Biosciences). Gates for flow cytometric acquisition and analyses were based on “fluorescence-minus-one” (FMO) controls and single stain compensation controls.
3
Multi-parameter Immunophenotyping of BMMCs
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Immunofluorescence surface staining was performed by adding a panel of directly conjugated Abs to BMMCs. After surface staining, cells were permeabilized using the Cytofix/Cytoperm kit (BD Pharmingen), and incubated with intracellular Abs. Labeled cells were measured using a FACSCanto II (BD Biosciences). Data were analyzed using Flowjo software. The following labeled Abs were used: IL-7Rα-PE (hIL-7Rα-M21), CD8-PeCy7 (RPA-T8), CCR7-FITC (150503), and CD28 BV421 (CD28.2) purchased from BD, CD122 (IL-2/IL-15Rβ)-APC (TU-27), CD45RA-PerCp (HI100), and KLRG-1-PeCy7 (2F1/KLRG1) purchased from Biolegend, CD3-APC-eFluor 780 (SK7) purchased from eBioscience.
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