The isolated ASCs' surface antigen expression was analyzed by flow cytometry according to a previously reported method [23] (link). One million passage 3 ASCs were suspended in 100 μL PBS containing 10 μg/ml fluorescein isothiocyanate-conjugated primary antibodies specific to mesenchymal stromal cells (MSCs) (CD29, CD44, CD90, and CD105) and hematopoietic cells (CD31 and CD45) (n = 3). The following expression markers reactive with the porcine antigen isoforms were used: Alexa Fluor 647 Mouse Anti-Pig CD29 (BD Bioscience, New Jersey, USA), Anti-CD44 antibody [IM7] (Abcam plc, Cambridge, UK), APC Mouse Anti-Human CD90 (BD-Biosciences), Anti-CD105 antibody [MEM-229] (Abcam plc), PE Mouse Anti-Rat CD31 (BD Biosciences), and Monoclonal Antibody to CD45/LCA (CD45R)-PE (Acris Antibodies, Inc. CA, USA) [24] (link), [25] (link), [26] (link), [27] (link), [28] (link). Cell fluorescence was evaluated with a Gallios flow cytometer (Beckman Coulter, Tokyo, Japan) and the data were analyzed using Karuza for Gallios software (Beckman Coulter).
Apc mouse anti human cd90
Manufactured by BD
Sourced in United Kingdom, United States
The APC mouse anti-human CD90 is a laboratory reagent used in flow cytometry applications. It is a monoclonal antibody that binds to the CD90 (Thy-1) cell surface antigen expressed on various cell types, including T cells, neurons, and stem cells. This antibody is conjugated with the fluorescent dye Allophycocyanin (APC), which enables the detection and identification of CD90-positive cells.
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Lab products found in correlation
Pe rat anti mouse cd31, by BD (1 mentions)
Gallios flow cytometer, by Beckman Coulter (1 mentions)
Stabilizing fixative, by BD (1 mentions)
Pe rat anti human cd184, by BD (1 mentions)
Facscanto 2 cytometer, by BD (1 mentions)
Cytofix cytoperm buffer, by BD (1 mentions)
Apc anti human cd31 antibody, by BioLegend (1 mentions)
Apc anti mouse cd140b antibody, by BioLegend (1 mentions)
Apc goat anti mouse igg, by BioLegend (1 mentions)
Facsaria fusion, by BD (1 mentions)
Facsdiva software, by BD (1 mentions)
Accutase cell dissociation reagent, by Thermo Fisher Scientific (1 mentions)
Apc mouse antihuman cd73, by BD (1 mentions)
Apc mouse igg1, by BD (1 mentions)
Clone mopc 21, by BD (1 mentions)
Apc mouse anti human cd13, by BD (1 mentions)
Apc mouse anti human cd105, by BD (1 mentions)
Apc mouse anti human cd44, by BD (1 mentions)
Moflo high speed cell sorter, by Beckman Coulter (1 mentions)
Fitc mouse anti human cd31, by BD (1 mentions)
6 protocols using apc mouse anti human cd90
1
Flow Cytometric Analysis of Porcine ASCs
2
Liver Cell Surface and Intracellular Staining
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For cell surface staining, liver cells were first incubated with DPBS-bovine serum albumin (BSA) 1.5% for 20 minutes at 4°C to prevent nonspecific binding. Next, the cells were washed with DPBS-BSA 1.5% and stained with 5 μL of PE rat anti-human CD184, APC mouse anti-human CD90, or their respective isotypes (BD Biosciences) for 30 minutes on ice. Finally, the cells were washed and fixed using a stabilizing fixative (BD Biosciences). For intracellular staining, liver cells were fixed and permeabilized with 200 μL of cytofix/cytoperm buffer (BD Biosciences) for 20 minutes at 4°C. The cells were then washed with perm/wash buffer and stained with PE rat anti-human CD184 or its isotype diluted in perm/wash for 30 minutes on ice. Next, the cells were washed twice and fixed with stabilizing fixative (BD Biosciences). Fluorescence was measured with a BD FACSCanto II cytometer on 10,000 cells using the FACSDiva software. Data analyses were performed with FlowJo software.
3
Cardiomyocyte FACS Sorting and Characterization
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Differentiated EBs were dissociated the same way as in the high throughput screening and suspended in 2% FBS/PBS. We analyzed and sorted the CMs using FACSAria Fusion (BD Biosciences), FACSDiva software (BD Biosciences) and FlowJo software. For Ki-67 staining, we used purified mouse anti-Ki-67 Clone B56 (RUO) (BD Biosciences, 556003, 1:200 dilution) and APC goat anti-mouse IgG (minimal x-reactivity) (Biolegend, 405308, 1:500 dilution). To sort the CMs derived from 1390C1 or 409B2, we used PE/Cyanine7 anti-human CD172a/b (SIRPα/β) antibody (BioLegend, 323808, 1:500 dilution), APC mouse anti-human CD90 (BD, 559869, 1:1000 dilution), APC anti-human CD31 antibody (BioLegend, 303116, 1:500 dilution), Alexa Fluor® 647 anti-human CD49a antibody (BioLegend, 328310, 1:500 dilution) and APC anti-mouse CD140b antibody (BioLegend, 136008, 1:500 dilution).
4
Retinal Organoid Immunosuppression Profiling
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After 30-45 days of induction, retinal organoids were cultured adherently with either RAP (20 μg/ml) or DEX (170 μg/ml) for 2 weeks; positive controls were treated without immunosuppressants. The tissues were then digested into single cells using an Accutase™ Cell Dissociation Reagent (A1110501, Gibco) as per the manufacturer's instructions. The cell suspensions obtained were incubated with APC mouse anti-human CD90 (561971, BD Biosciences, USA) for 25 min. Flow cytometry was carried out using an LSRFortessa flow cytometer (BD Biosciences), and the data obtained were analyzed using the FlowJo software (Tree Star Inc., USA). All experiments were replicated three times.
5
Surface Marker Analysis of Mesenchymal and Cancer Cells
6
Flow Cytometry Analysis of Cell Subpopulations
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Flow cytometry scans were performed to evaluate the evolution of each cell subpopulation with time. To do this, at each selected time points (2 and 7 days), cells in 5 replicates per group were trypsinized, pooled, and subsequently blocked for 30 min with BSA 1% in DPBS. As suggested by the supplier, a minimum of 1 x 10 6 cells per group were incubated in a 100-µl test sample for 30 min at 4°C with FITC mouse anti-human CD31 (1:5, BD Bioscience, 560984) and APC mouse anti-human CD90 (1:20, BD Bioscience, 561971) diluted in PBSA. Finally, cells were rinsed twice, re-suspended with DPBS and scanned in a High Speed Cell Sorter MoFlo flow cytometer (Beckman-Coulter, CA, USA).
1:300 propidium iodide (1 mg/mL in water, Sigma-Aldrich) was added to the cell suspension to discard dead cells from the analyses.
1:300 propidium iodide (1 mg/mL in water, Sigma-Aldrich) was added to the cell suspension to discard dead cells from the analyses.
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