Tumors were resected from mice, dissociated by collagenase and hyaluronidase (StemCell Technologies, Vancouver, BC, Canada), incubated in ACK red blood cell lysis buffer (Invitrogen), and filtered through a sterile 40 µm cell strainer. ALDH enzymatic activity was stained using Aldefluor Kit (StemCell Technologies) or AldeRed ALDH Detection Assay (MilliporeSigma, Burlington, MA, USA). Briefly, 1 × 106 cells were incubated with activated ALDH substrate or the equivalent volume of ALDH inhibitor diethyl aminobenzaldehyde (DEAB). DEAB controls were included for all treatment conditions. Cells were rinsed with PBS and stained for CD44 with either CD44-PE or CD44-APC (R&D Systems, Minneapolis, MN, USA) for 15 min at 4 °C. Human cells were identified by anti-HLA-ABC (PE; BD Pharmingen, NJ, USA). Viable cells were stained with DAPI (Molecular Probes, Eugene, OR, USA). For cell sorting, ALDHhighCD44high CSC population was sorted against the remaining bulk tumor cells (i.e., ALDHhighCD44low, ALDHlowCD44high, and ALDHlowCD44low). All flow cytometry analyses were conducted in a BD LSRFortessa flow cytometer (BD Biosciences). Results were analyzed with FlowJo software (LLC; Ashland, OR, USA) in triplicate wells per condition.
Anti hla abc pe
Manufactured by BD
Sourced in United States
Anti-HLA-ABC-PE is a monoclonal antibody conjugated with the fluorescent dye phycoerythrin (PE). It is used for the detection and quantification of HLA-ABC (human leukocyte antigen class I) molecules on the surface of cells by flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Anti hla dr pe, by BD (4 mentions)
Anti cd83 pe, by BD (3 mentions)
Anti cd123 apc, by Miltenyi Biotec (2 mentions)
Anti cd80 pe, by BD (2 mentions)
Facscalibur, by BD (2 mentions)
Ack red blood cell lysis buffer, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Cd44 pe, by R&D Systems (1 mentions)
Cd44 apc, by R&D Systems (1 mentions)
Lsrfortessa flow cytometer, by BD (1 mentions)
Aldefluor kit, by STEMCELL (1 mentions)
Collagenase and hyaluronidase, by STEMCELL (1 mentions)
Anti cd40 fitc, by BD (1 mentions)
Cellgro gmp dc medium, by CellGenix (1 mentions)
Anti ccr7 apc, by Miltenyi Biotec (1 mentions)
Anti cd80 apc, by BD (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Cellquest pro software, by BD (1 mentions)
Facscalibur cytometer, by BD (1 mentions)
Anti cd14 fitc, by BD (1 mentions)
6 protocols using anti hla abc pe
1
Isolation and Characterization of Cancer Stem Cells
2
Characterization of moDC and mDC Responses
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6×105 mDCs or moDCs were cultured for 18 h at 37°C in 200 µL CellGro GMP DC medium (CellGenix, Cat. No.: 20801-0500) supplemented with 50 µg/mL AfuLy, 1-5 µg/mL Aspergillus proteins CcpA or Shm2, 1 µg/mL LPS (Sigma, positive control), or plain medium (negative control). Thereafter, DCs were stained with anti-CD1a-APC (moDCs) or anti-CD1c-APC (mDCs), anti-CD14-FITC, anti-CD80-APC, anti-CD83-PE, anti-CD86-FITC, anti-CD40-FITC, anti-HLA-ABC-PE, anti-HLA-DR-PE (BD), and anti-CCR7-APC (Miltenyi Biotec) in three different panels. Samples were analyzed using a FACS Calibur cytometer (BD), Cell Quest Pro software (BD), and Flow Jo software (version 10.6.2.). Dead cells were excluded from analysis by light scatter (FSC/SSC) properties.
3
Immunophenotypic Characterization of EMT
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The following antibodies were used to characterise cancer cell immunophenotype before and after EMT induction: anti-CD324-PE (Biolegend, San Diego, CA, USA), anti-Vimentin (R&D System, Minneapolis, MN, USA), anti-HLA-A,B,C-PE (BD Bioscience, San Jose, CA, USA), anti-CD54-PE (BD Bioscience), anti-HLA-DR-PE (BD Bioscience), and corresponding isotype controls. Cells were stained according to the manufacturer's instructions and analysed by FACSCanto II (BD Bioscience). For immunofluorescence, cancer cells were immunostained with the following antibodies: E-cadherin (Abcam, Cambridge, MA, USA), Vimentin (Dako, Glostrup, Denmark), relative secondary antibody (Jackson Immunoresearch, West Grove, PA, USA), and Topro3 (Invitrogen) for nucleus counterstaining. Images were acquired with the Zeiss LSM 510 (Zeiss, Oberkochen, Germany) confocal microscope.
4
Phenotypic Analysis of DC Maturation
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Phenotypical assessment of DC maturation after 48 h of co-culture with tumor cells was performed by flow cytometry. Briefly, cells were washed in PBA, incubated with Fc-receptor blocking buffer (2% HS in PBS, 15 min at 4°C) and subsequently stained with primary antibodies in PBA (30 min at 4°C). Monoclonal directly labeled anti-human antibodies used were (Table S1): anti-CD11c-APC (Miltenyi biotec, clone MJ4-27G12, catalog# 130-092-412), anti-CD123-APC (Miltenyi biotec, clone AC145, catalog# 130-090-901), anti-CD80-PECy7 (BD PharMingen, clone L307.4, catalog# 561135), anti-CD86-PECy7 (BD PharMingen, clone 2331, catalog# 561128), anti-HLA-ABC-PE (BD PharMingen, clone G46-2.6, catalog# 555553), and anti-HLA-DR-PE (BD PharMingen, clone G46-6, catalog# 555812). Appropriate isotype controls were included. Geometric mean fluorescence intensity (GeoMFI) of maturation markers was assessed on CD11c+ (for CD1c+ and CD16+ DCs) or CD123+ (for pDCs) populations. As a positive control, DCs were stimulated with poly I:C (CD1c+ and CD16+ DCs, 2 µg/mL, Enzo Life Science, catalog# ALX-746-021-M005) or R848 (pDCs, 4 µg/mL, Enzo Life Science, catalog# ALX-420-038-M025). To improve in vitro pDC viability, IL-3 (10 ng/mL, Cellgenix, catalog# 1002-050) was added to culture medium.
5
Phenotypic Analysis of PLGA Particle Uptake
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Uptake of PLGA particles and the phenotype of mDCs and pDCs were determined by flow cytometry. Plasmacytoid DCs and mDCs were cultured overnight with different concentrations of PLGA particles containing PFC and atto-647. The following primary monoclonal antibodies (mAbs) and appropriate isotype controls were used: anti-BDCA2-PE, anti-CD123-APC, anti-CD11c-PE and anti-BDCA-1-APC (all Miltenyi Biotec); anti-HLA-ABC-PE, anti-HLA-DR/DP-FITC, anti-CD80-PE, anti-CD83PE, anti-CD86-APC (all BD Bioscience Pharmingen, CA, USA); anti-CD40-PE (Beckman Coulter, Mijdrecht, The Netherlands). Cells were analyzed by flow cytometry on a FACSCalibur (BD Biosciences, CA, USA).
6
Phenotypic Characterization of mDCs
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The purity of mDCs after CliniMACS isolation and the phenotype of the mDCs were determined by flow cytometry. The following primary mAbs and the appropriate isotype controls were used: anti-CD1b/c-FITC (Diaclone), anti-CD20-PE, anti-CD45-PerCP, anti-CD14-APC, anti-HLA-DQ-PE (all BioLegend), anti-HLA-ABC-PE, anti-HLA-DR-PE, anti-CD80-PE, anti-CD83-PE, and CD86-PE (all BD Pharmingen). Flow cytometry was performed with a FACSCalibur (BD Biosciences).
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