WJ-MSCs and HUVECs, respectively, at the eighth and the sixth passage, were treated with 0.05% trypsin–EDTA and collected; 106 cells per sample were incubated with 1 μg of the specific antibody, conjugated with fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), phycoerythrin-cyanine 5.5 (PE Cy5.5), or Alexa Fluor 488 for 30 min at 4°C in the dark. WJ-MSCs were stained using the following antibodies: anti-CD31, anti-CD73, anti-CD13, anti-CD90, anti-CD117, anti-CD14, anti-CD34, anti-CD105, anti-CD146, anti-CD133, anti-CD144, anti-ESA, anti-HLA-ABC, anti-HLA-DR, anti-CD45 (Becton Dickinson [BD], San Jose, CA), anti-CD29, anti-CD44, and anti-CD166 (Ancell, Bayport, MN). HUVECs were stained with anti-CD146 (BD) and anti-CD144 (Acris Antibodies, San Diego, CA). After incubation, cells were washed and acquired with a flow cytometer (FACS Calibur; BD), collecting 10,000 events per sample. Data were analyzed by the FlowJo software v8.8.6 (TreeStar, Ashland, OR). The mean fluorescence intensity (MFI) ratio values were calculated (i.e., dividing the MFI of positive events by the MFI of negative events).22 (link),23 (link)
Anti cd146
Manufactured by BD
Sourced in Germany, United States
Anti-CD146 is a laboratory reagent used for the detection and identification of CD146, a cell surface marker. CD146 is expressed on various cell types, including endothelial cells, smooth muscle cells, and some types of cancer cells. The Anti-CD146 reagent can be used in flow cytometry and other analytical techniques to study the presence and distribution of CD146-positive cells in samples.
Automatically generated - may contain errors
Lab products found in correlation
Facscalibur, by BD (3 mentions)
Anti cd90, by BD (3 mentions)
Anti cd73, by BD (3 mentions)
Anti hla dr, by BD (3 mentions)
Anti cd45, by BD (2 mentions)
Anti cd34, by BD (2 mentions)
Anti cd144, by OriGene (2 mentions)
Anti cd105, by BD (2 mentions)
Anti cd117, by BD (2 mentions)
Anti cd31, by BD (1 mentions)
Anti cd13, by BD (1 mentions)
Anti oct3 4, by BD (1 mentions)
Anti ssea4, by BD (1 mentions)
Anti sox2, by BD (1 mentions)
Anti cd34, by Beckman Coulter (1 mentions)
Stempro differentiation kit, by Thermo Fisher Scientific (1 mentions)
Cellquest software, by BD (1 mentions)
Facscalibur sorter, by BD (1 mentions)
Anti cd9, by BD (1 mentions)
Anti cd81, by BD (1 mentions)
4 protocols using anti cd146
1
Flow Cytometric Characterization of MSCs and HUVECs
2
Characterization of hPDLSCs by Flow Cytometry
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hPDLSCs at the second passage were collected; 5 × 105 cells per sample were incubated with 1 μg of the specific antibody, conjugated with fluorescein isothiocyanate, phycoerythrin, allophycocyanin, phycoerythrin-cyanine 5.5, or Alexa Fluor 488 for 30 min at 4 °C in the dark. hPDLSCs were stained using the following antibodies: anti-CD13, anti-CD29, anti-CD44, anti-CD45, anti-CD105, anti-CD166 (Ancell, MN, USA), anti-CD14, anti-CD133 (BergischGladbach, Germany), anti-CD73, anti-CD90, anti-CD117, anti-CD146, anti-CD271, anti-Sox2, anti-HLA-DR, anti-SSEA4, anti-OCT3/4 (Becton Dickinson, BD, San Jose, CA, USA), anti-CD144 (Acris Antibodies, Herford, Germany) and anti-CD34 (Beckman Coulter, Fullerton, CA, USA). After incubation, cells were acquired with a flow cytometer (FACS Calibur; BD). Data were analyzed by the FlowJo software v8.8.6 (TreeStar, Ashland, OR, USA) [17 (link)].
3
Characterization of hMSCs and Extracellular Vesicles
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The StemPro Differentiation Kit (Life Technologies) was used according to the manufacturer’s recommendations to determine the capacity of hMSCs to undergo adipogenic or chondrogenic differentiation. Characterization of hMSCs and hMSC-microvesicles was performed by fluorescence-activated cell sorting (FACS) utilizing a FACSCalibur™ sorter (BD Biosciences) and analyzed by CellQuest software (BD Biosciences). FITC-conjugated antibodies (anti-CD73, anti-CD90, anti-CD105, anti-CD146, anti-CD14, anti-CD34, anti-CD45, and anti-HLA-DR; BD Biosciences) were incubated with samples for 30 min in PBS containing 0.5% BSA at 4 °C. Mouse isotypic IgG was used as a control.
FACS analysis of hMSC-exosomes was performed using exosome capture beads based on anti-CD63 coupled antibody (Invitrogen Dynabeads Exosome Human CD63 Isolation/Detection Reagent) following the manufacturer’s recommendations. Specific FITC-conjugated exosomal markers (anti-CD9 and anti-CD81; BD Biosciences) were used as above.
FACS analysis of hMSC-exosomes was performed using exosome capture beads based on anti-CD63 coupled antibody (Invitrogen Dynabeads Exosome Human CD63 Isolation/Detection Reagent) following the manufacturer’s recommendations. Specific FITC-conjugated exosomal markers (anti-CD9 and anti-CD81; BD Biosciences) were used as above.
4
Establishment and Characterization of Human Melanoma Cell Lines
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Human melanoma samples were obtained from patients selected at the IRCCS Ospedale Policlinico San Martino under local Institutional-Review-Board-approved protocols (CER Liguria: 046REG2017). Patient biopsies were placed in a sterile conical tube containing NaCl 0.9% and 1% antibiotic and antimycotic on wet ice and handled no later than 4 h after resection. Upon arrival, biopsies were manually minced using a sterile scalpel and dissociated using 70 µm cell strainers (Miltenyi Biotec) at room temperature. Cells were cultured in RPMI 10%FBS, 1%HEPES, 1%Na Pyruvate, 2%Glutamine, 1%Pen/Strep, and 1% antimycotic and stabilized as culture after the 8th passage. Positivity for melanoma antigens was assessed by flow cytometry (FACSCalibur; Becton Dickinson, East Rutherford, NJ, USA) in Mel_3 and Mel_15 cells using anti-CD146 (BD Pharmingen, cod 550315) and anti-MCSP (Miltenyi Biotec, cod 130-091-225) monoclonal antibodies.
A549, a NSCLC cell line expressing functional ATM, was purchased from the ICLC cell bank of IRCCS Ospedale Policlinico San Martino and cultured in RPMI 10%FBS, 2%Glutamine, and 1%Pen/Strep.
A549, a NSCLC cell line expressing functional ATM, was purchased from the ICLC cell bank of IRCCS Ospedale Policlinico San Martino and cultured in RPMI 10%FBS, 2%Glutamine, and 1%Pen/Strep.
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