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.
Cd14 pecy7 m 5e2
Manufactured by BD
CD14-PECy7 (M-5E2) is a monoclonal antibody that binds to the CD14 antigen, which is expressed on the surface of monocytes and macrophages. The antibody is conjugated with the Peridinin-Chlorophyll-Protein Complex (PECy7) fluorescent dye, which can be detected using flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Live dead fixable aqua stain, by Thermo Fisher Scientific (1 mentions)
Cd8 v500 rpa t8, by BD (1 mentions)
Cd3 apc efluor780 sk7, by Thermo Fisher Scientific (1 mentions)
Formaldehyde, by Merck Group (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
Emp1 fitc, by Calibre Scientific (1 mentions)
Human fc blocking reagent, by Miltenyi Biotec (1 mentions)
Cd56 pecy7 b159, by BD (1 mentions)
Cd19 apc sj25c1, by BD (1 mentions)
7 aad, by BD (1 mentions)
Aria 2, by BD (1 mentions)
2 protocols using cd14 pecy7 m 5e2
1
PBMCs Phenotyping and Intracellular Staining
2
Multiparameter PBMC Immunophenotyping for scRNAseq
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For isolation of cells for scRNAseq, freshly isolated PBMC were FC block- treated (2 ul FC blocking reagent human (Miltenyi Biotech) and 48 ul staining buffer (1% BSA in PBS, filtered through a 40 um filter)/1x106 cells) and incubated 10’ on ice. Cells were washed and stained in 50 ul final/1x106 cells of antibody cocktail (CD3-APC (HIT3α, BD), CD19-APC (SJ25C1, BD), CD14-PE-Cy7 (M5E2, BD), CD56-PE-Cy7 (B159, BD), HLA-DR-APC-H7 (B159, BD), CD11c-AF700 (3.9, eBioscience), CD123-BV650 (6H6, Biolegends), CD16-BV605 (3G8, Biolegends)). Life- dead staining with 7AAD (BD) was done shortly before sorting on a FACS (ARIAII, BD) with the 100 uM nozzle using gating strategy shown in Supplemental Figure 1 Supplemental Figure 4A
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!