The following fluorochrome-conjugated monoclonal antibodies were used in this study: CD3, CD4, CD8, CD19, and IFNγ from Beckman Coulter (Indianapolis, IN); CD71, HLA-A2, and HLA-A, B, C from BioLegend (San Diego, CA); CD95 (Fas) and CD107a from BD Biosciences (San Jose, CA); and CD34 (QBEnd-10) from Abnova (Taipei, Taiwan). Cell viability was assessed using 7-amino actinomycin D (7-AAD) (BD Biosciences) staining. We used the Gallios Flow Cytometer (Beckman Coulter) to acquire flow cytometric data and Kaluza Analysis Software (Beckman Coulter) to analyze data and for graphical representation.
Hla abc
Manufactured by BioLegend
Sourced in United States
HLA-ABC is a lab equipment product that detects and quantifies the expression of human leukocyte antigen (HLA) class I molecules. It provides a tool for researchers to investigate the role of HLA class I in various biological processes and disease states.
Automatically generated - may contain errors
Lab products found in correlation
Hla dr, by BioLegend (6 mentions)
Cd105, by BioLegend (3 mentions)
Pd l1, by BioLegend (2 mentions)
Cytokeratins 18 and 19, by BioLegend (2 mentions)
α smooth muscle actin, by R&D Systems (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Cyan adp analyzer, by Beckman Coulter (2 mentions)
Facscalibur, by BD (2 mentions)
Fitc labeled annex in 5 antibody, by Thermo Fisher Scientific (2 mentions)
Apc conjugated mabs against human β2m, by BioLegend (2 mentions)
7 aminoactinomycin d 7 aad, by BD (1 mentions)
Gallios flow cytometer, by Beckman Coulter (1 mentions)
Ifn γ, by Beckman Coulter (1 mentions)
Kaluza analysis software, by Beckman Coulter (1 mentions)
Interleukin 2 (il 2), by BD (1 mentions)
Dc sign, by BD (1 mentions)
Accuri c6 cytometer, by BD (1 mentions)
Sr b1, by Novus Biologicals (1 mentions)
Sr a1, by R&D Systems (1 mentions)
Tnf α, by Beckman Coulter (1 mentions)
24 protocols using hla abc
1
Multiparameter Flow Cytometry Analysis
2
Endocytic receptor and immune cell analysis
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Endocytic receptors were stained using the following antibodies: CD206/MR (eBioscience), DC-SIGN, CD36 (both from BD Biosciences), SR-A1 (R&D Systems), LOX-1 (BioLegend), and SR-B1 (Novus Biologicals). DC and T cell phenotypes were examined using antibodies against the following markers: HLA-ABC (BioLegend), CD206, CD40, CD80, CD83, IL-2 (BD Biosciences), CD4, CD8, TNF-α, IL-2, and HLA-DR (Beckman Coulter). Data were acquired with an Accuri C6 cytometer (BD Biosciences) and analyzed using CFlow Plus software.
3
Immunogenicity of PBNP-PTT Assessed
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Antibodies were purchased from Biolegend (San Diego, CA) and Abcam (Cambridge, UK). After 24 h incubation at 37 °C post-PBNP-PTT, cells were harvested and stained with Zombie Aqua Fixable viability dye (Biolegend, #423102), blocked with human TruStain Fc block (Biolegend, #422302), and stained with fluorescent antibodies against calreticulin (Abcam, #ab83220), CD80 (Biolegend, #305238), CD86 (Biolegend, #305428), PD-L1 (Biolegend, #329714), B7-H3 (Biolegend, #351010), HLA-ABC (Biolegend, #311432), HLA-DR (Biolegend, #307633), PVR (Biolegend, #337628), and GD2 (Biolegend, #357308). Flow cytometry was performed using the Cytek Aurora cytometer (Cytek Biosciences, Fremont, CA, USA), and cytometric analysis was done using FlowJo software (Ashland, OR, USA) to assess the immunogenicity correlates described above as a function of PBNP-PTT thermal dose.
4
Isolation and Characterization of Patient-Derived Cell Lines
5
Quantifying Endothelial Cell Surface Proteins
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Surface protein expression was quantified using flow cytometry. Briefly, endothelial cells were harvested, washed in 1% BSA in PBS, and incubated with an antibody against HLA-DR (clone L243, Biolegend) for 30 min on ice. After antibody incubation, endothelial cells were washed three times using 2% BSA in PBS and analyzed on a Attune NxT Flow Cytometer. Other endothelial cell antibodies used for flow cytometry include HLA-A,B,C (Biolegend) and CD31 (Biolegend). All results were analyzed using the FlowJo software (FlowJo LLC).
6
Phenotypic Characterization of Human Umbilical Cord MSCs
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Surface markers for human umbilical cord MSCs were quantified by flow cytometry using CD13, CD14, CD29, CD31, CD34, CD44, CD45, CD56, CD59, CD61, CD71, CD105, CD106, CD133, HLA-ABC, HLA-DR (all from BioLegend), CD73 (BD Pharmingen), and CD90 (Serotec) antibodies. MSCs (5×105 cells) were washed twice with PBS, resuspended in 100 μl of PBS containing monoclonal antibodies, and incubated for 30 min at 4°C. These cells were then washed twice and resuspended in 500 μl of PBS. Fluorescence analysis was performed with a flow cytometer (FACS Caliber, BD). The non-specific binding of the fluorescein isothiocyanate (FITC) and phosphatidyl ethanolamine (PE) conjugates were determined in control samples using a mouse IgG1-FITC and IgG1-PE negative control (Serotec). Analysis was conducted using the WinMDI 2.9 software.
7
Flow Cytometry Analysis of EndoC-βH1 Cells
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EndoC-βH1 cells were trypsinized, washed three times in Hanks' balanced salt solution (HBSS)/1% BSA and incubated at 4 °C for 15 min in the dark with the following antibodies: LDLR (1/100 dilution; catalog no.: MAB CL 472413; Fisher scientific), CD36 (1/10 dilution; catalog no.: 555455; BD Pharmingen), PDL1 (1/100 dilution; catalog no.: 329714; BioLegend), HLA-ABC (1/100 dilution; catalog no.: 3114410; BioLegend) diluted in HBSS medium/1% BSA (Gibco/Roche Diagnostics). Fluorochrome-conjugated primary antibodies were used with the exception of LDLR detection, for which a step of incubation with Alexa-Fluor 488 (1/400 dilution; Invitrogen) secondary antibody was performed. Following rinsing in HBSS/1% BSA and incubation with FACS medium containing propidium iodide (1/4000 dilution; Sigma–Aldrich), analysis was carried out using an FACS Aria III (BD Biosciences). Dead cells were excluded from analyses by using propidium iodide. Data were analyzed using FlowJo 10.7 software (Research Resource Identifier: SCR_008520; BD Life Sciences). Results are expressed in mean fluorescence intensity fold changes relative to the control condition.
8
Immunofluorescence Analysis of PCa Cells
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Cells were fixed and permeabilized with Perm/Wash Buffer (Cat. # 554723, BD Biosciences). Samples were blocked with Image-iT FX signal enhancer for 30 min and incubated for 2 hours at room temperature with primary antibodies combined with reagents of Zenon Alexa Fluor 488 (green) or 555 (red) labeling kit (Invitrogen). To detect Ki-67 in PCa cells co-cultured with MC3T3-E1 cells in vitro, human Ki-67 (cat. # ab15580, Abcam) and HLA-ABC (Cat. # 311402, BioLegend) antibodies were used as primary antibodies. To detect Ki-67 in PCa cells in bone marrow sections, human Ki-67 (cat. # ab15580, Abcam) and pan cytokeratin (cat. # ab867364, Abcam) antibodies were used as the primary antibody. To detect human TGFBR2 and TGFBR3, human TGFBR2 (cat. # ab61213, Abcam) and human TGFBR3 (cat. # ab78421, Abcam) antibodies were used as the primary antibody. After washing with PBS, the slides were mounted with ProLong Gold antifade reagent with DAPI (Invitrogen). Images were taken with Nikon A-1-B confocal microscope.
9
Immunophenotypic Characterization of hWJSCs
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Cultured hWJSC monolayers were first disassociated using trypsin (TrypLE™ Express, Thermo Scientific) for 3–5 min at 37 °C in a 5% CO2 in air, then centrifuged and washed in phosphate buffered saline (PBS) and blocked with 10% normal goat serum (NGS) (Thermo Scientific) for 10 min at room temperature to prevent non-specific binding following manufacturer’s instruction. The cells were then incubated with mouse monoclonal primary antibodies for a series of CD markers viz., CD14, CD19, CD29, CD34, CD44, CD45, CD73, CD90, CD105, HLA-ABC, and HLA-DR (1:100) (Biolegend, San Diego, CA) for 30 min at 4 °C. This was followed by incubation with Alexa Fluor®488 (1:5000) goat anti-mouse secondary antibody (Thermo Scientific) for 30 min at 4 °C in the dark [29 (link)]. The cells were finally washed in PBS (−), re-suspended in 10% NGS, and filtered using a 40-μm nylon strainer (BD) to remove any cell clumps and then analyzed using a CyAn™ ADP Analyzer (Beckman Coulter, Fullerton, CA).
10
Isolation and Characterization of Patient-Derived Cells
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