Flow cytometric analysis was performed on FAPs and SCs using PDGFRα-Alexa Fluor (AF) 647 (1:50; Abcam, ab270085), ITGA5-APC (1:50; Miltenyi Biotec, 130-110-591), CD14-APC (1:50; Miltenyi Biotec, 130-110-578), CD61-APC (1:50; Miltenyi Biotec, 130-110-887), combined with CD56-PE (1:10; BD Biosciences, 345812) antibodies on a MACSQuant10 flow cytometer (Miltenyi Biotec). Unstained cells were used routinely as negative controls and to define gating parameters. Flow cytometric data was analysed using FlowJo (FlowJo LLC, version 10.7.1).
Cd14 apc
Manufactured by Miltenyi Biotec
Sourced in United States, Germany
CD14-APC is a fluorochrome-conjugated antibody that specifically binds to the CD14 surface antigen. CD14 is a glycosylphosphatidylinositol (GPI)-anchored protein that serves as a co-receptor for the detection of lipopolysaccharide (LPS) on the surface of monocytes and macrophages. The APC (Allophycocyanin) fluorochrome is attached to the CD14 antibody, allowing for the detection and analysis of CD14-positive cells using flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Cd34 pe, by Miltenyi Biotec (2 mentions)
Cd56 pe, by BD (1 mentions)
Macsquant 10, by Miltenyi Biotec (1 mentions)
Ab59776, by Abcam (1 mentions)
Cd31 pe, by BD (1 mentions)
Cd31 fitc, by BD (1 mentions)
Oct4 c 10, by Santa Cruz Biotechnology (1 mentions)
Mouse anti human cd45 fitc, by Miltenyi Biotec (1 mentions)
Cd15 fitc, by Miltenyi Biotec (1 mentions)
Lamp2, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Cd45 apc, by BD (1 mentions)
Cd11c percp cy5, by BioLegend (1 mentions)
Cd16 v500, by BD (1 mentions)
Fixable viability dye, by Thermo Fisher Scientific (1 mentions)
Facscanto 2, by BD (1 mentions)
Hla dr fitc, by BD (1 mentions)
Cd34 fitc, by BioLegend (1 mentions)
Hla dr apc, by BD (1 mentions)
Facs lsrfortessa, by BD (1 mentions)
8 protocols using cd14 apc
1
Flow Cytometric Analysis of FAPs and SCs
2
Comprehensive Antibody Panel for Cell Analysis
3
Multicolor Flow Cytometry Profiling of PBMCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PBMCs were stained with fixable viability dye (for dead cell exclusion, eBioscience, San Diego, CA, USA) and antibodies for CD14 APC (Miltenyi Biotec), CD11c PerCPCy5.5 (Biolegend, San Diego, CA, USA), HLA-DR FITC (BD Biosciences, Franklin Lakes, NJ, USA), CD16 V500 (BD Biosciences), Tie2 PE and its respective isotype control (R&D systems). Data were acquired on a BD FACSCanto II (BD Biosciences). After excluding debris, doublets, and dead cells, cell populations (see gating strategy in Table 2 ) were analyzed using the FlowJo software (Tree Star, Ashland, OR, USA). Results were expressed as percentage of positive cells.
4
Immunophenotyping of Human Mesenchymal Stem Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HMSCs were harvested by scrapping or trypsinization from T175 cm2 flasks. Then, the cells were incubated in staining buffer [2% FBS in phosphate-buffered saline (PBS)], stained with a mouse anti-human CD146–Alexa Fluor 647 (clone P1-H12, BD Biosciences), a mouse anti-human CD31–Alexa Fluor 488 (BD Biosciences, San Jose, CA) antibody, a mouse anti-human CD105–Alexa Fluor 647 (BD Biosciences, San Jose, CA), a mouse anti-human CD90–fluorescein isothiocyanate (FITC) and a mouse anti-human CD73–allophycocyanin (APC) (Miltenyi Biotec, Germany), a CD14-APC (Miltenyi Biotec), a CD34-FITC (BioLegend), and an HLA-DR–APC (BD Biosciences).
The percentages of CD73-, CD90-, CD105-, CD146-, CD31-, CD34-, and HLA-DR–positive cells were analyzed using a FACS LSRFortessa (BD Biosciences, San Jose, CA) or an ImageStream (Amnis) flow cytometer. To validate the specificity of the antibody staining, the distributions of fluorescently labeled cells were compared to cells stained with isotype controls: mouse immunoglobulin G1 (IgG1), k-PE-Cy5 (clone MOPC-21, BD Biosciences), and mouse IgG2a K isotype control FITC (BD Biosciences, San Jose, CA). Alternatively, HMSCs were sorted on the basis of their level of expression of CD146 or their size [forward scatter (FSC) and side scatter (SSC)] using a FACSAria III (BD Biosciences, San Jose, CA).
The percentages of CD73-, CD90-, CD105-, CD146-, CD31-, CD34-, and HLA-DR–positive cells were analyzed using a FACS LSRFortessa (BD Biosciences, San Jose, CA) or an ImageStream (Amnis) flow cytometer. To validate the specificity of the antibody staining, the distributions of fluorescently labeled cells were compared to cells stained with isotype controls: mouse immunoglobulin G1 (IgG1), k-PE-Cy5 (clone MOPC-21, BD Biosciences), and mouse IgG2a K isotype control FITC (BD Biosciences, San Jose, CA). Alternatively, HMSCs were sorted on the basis of their level of expression of CD146 or their size [forward scatter (FSC) and side scatter (SSC)] using a FACSAria III (BD Biosciences, San Jose, CA).
5
Investigating PBMC Activation in Sepsis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Peripheral blood mononuclear cells (PBMCs) were purified from blood samples of septic patients and healthy donors by density gradient centrifugation using lymphocytes isolation solution (Rafer, Zaragoza, Spain). PBMCs were counted and 5 million per condition were cultured in T25 flasks in Roswell Park Memorial Institute (RPMI) Medium 1640 + GlutaMAX™ (Gibco, Life Technologies, CA, USA) containing 2% Fetal Bovine Serum (FBS), 100 units/mL penicillin, and 100 mg/mL streptomycin, for 4 hours, with or without LPS (10 ng/ml from E. coli O111:B4, Sigma-Aldrich, Darmstadt, Germany). Cells were collected and stained with CD14 (APC) (Miltenyi Biotec, ref: #130-091-243), CD15 (FITC), and pSTAT1 (mouse Anti-pStat1 BV421 (pY701), BD Biosciences; ref: #562985), using mouse IgG2a, κ Isotype BV421 (BD Biosciences; ref: #563464) as a control according to the BD Fixation/Permeabilization Solution Kit (#554714) and the antibodies manual.
6
Phenotyping Immune Cell Surface Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To study cell-surface markers, cells were collected using Versene, a non-enzymatic dissociation buffer (ThermoFisher). Cells were resuspended in the staining buffer (PBS with 4% fetal bovine serum and 2 mM ethylenediaminetetraacetic acid (EDTA)). Cells were then incubated in ice with Fc block reagent (Miltenyi Biotec) for 10 minutes, and stained with the viability dye LIVE/DEAD™ Fixable Violet (ThermoFisher), following the manufacturer's protocol.
Cells were then stained to study the proteins of interest, using the following antibodies: CD16 (APC) (#130-113-389, Miltenyi Biotec), CD14 (APC) (#130-110-520, Miltenyi Biotec), CD163 (FITC) (#33618, BioLegend), CD1a (PE) (#300106, BioLegend), CD80 (PE) (#H12208P, eBioScience), CD83 (APC) (#130-110-504, Miltenyi Biotec), CD86 (APC) (#130-113-569, Miltenyi Biotec), HLA-DR (PE) (#12-9956-42, eBioScience).
After staining, cells were fixed with PBS + 4% paraformaldehyde (Electron Microscopy Sciences) and analyzed within 2 days using a BD FACSCanto™ II Cell Analyzer (BD Biosciences). Data were analyzed with the FlowJo v10 software.
Cells were then stained to study the proteins of interest, using the following antibodies: CD16 (APC) (#130-113-389, Miltenyi Biotec), CD14 (APC) (#130-110-520, Miltenyi Biotec), CD163 (FITC) (#33618, BioLegend), CD1a (PE) (#300106, BioLegend), CD80 (PE) (#H12208P, eBioScience), CD83 (APC) (#130-110-504, Miltenyi Biotec), CD86 (APC) (#130-113-569, Miltenyi Biotec), HLA-DR (PE) (#12-9956-42, eBioScience).
After staining, cells were fixed with PBS + 4% paraformaldehyde (Electron Microscopy Sciences) and analyzed within 2 days using a BD FACSCanto™ II Cell Analyzer (BD Biosciences). Data were analyzed with the FlowJo v10 software.
7
Flow Cytometry Immunophenotyping Panel
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MACSQuant Analyzer, MACSQuant Analyzer VYB (Miltenyi Biotec), or FACSCanto, LSRII (Becton Dickinson, NJ) was used to analyze cell populations by flow cytometry. The following antibody and protein conjugates were used: CD3-PE, CD3-APC-Vio770, CD4-APC, CD4-VioGreen, CD8-APC-Vio770, CD8-VioGreen, CD14-APC, CD16-PE, CD19-APC, CD20-PerCP-Vio770, CD20-PE-Vio770, CD34-APC, CD34-PE, CD45-VioBlue, CD45-VioGreen, CD45RO-PE-Vio770, CD56-PE, CD62L-VioBlue, CD95-APC, CD45-VioBlue (mouse) (all from Miltenyi Biotec); ErbB2-Fc fusion protein (R&D Systems), anti-human-IgG (Fc gamma-specific) PE (eBioscience). 7AAD staining was used for dead cell exclusion and Violet Cell Trace (Thermo Fisher) for SupT1 cell tracking.
8
Multiparameter Flow Cytometry Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were washed twice with PBS and incubated in FACS-buffer (1% bovine serum albumin) with surface antibodies against CD71-VB405 (Miltenyi Biotec; 1:200), CD235-PE (Acris; 1:2500), CD14-APC (Miltenyi Biotec; 1:50), CD16-PE (BD Biosciences; 1:80); CD36-FITC (Pelicluster, Amsterdam, The Netherlands; 1:100); CD34-APC (IQ products, Groningen, the Netherlands; 1;10). Isotype controls were IgG1k-FITC (biolegends), IgG1-PE (Diaclone); IgG1k-APC (eBioscience). For hemoglobin staining on erythrocytes cells were fixed with 0.025% glutaraldehyde (sigma-Aldrich) and 0.5% paraformaldehyde (Signa-Aldrich) in PBS and permeabilized with 0.5% NP40 (Sigma-Aldrich) prior to staining HbA-PE (Santa Cruz; 1:1000) and HbF-APC (Invitrogen; 1:1000).
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!