To assess the effect of IFN-γ on the cell-surface phenotype of chordoma cells, cells were untreated or treated with 50 ng/mL of IFN-γ (R&D Systems, Minneapolis, MN) for 24 h. Cells were then harvested and stained with the following antibodies: HLA-ABC-FITC (BD Biosciences, San Jose, CA), PD-L1-APC (clone 29E.2A3; BioLegend, San Diego, CA), CD133-PE (Miltenyi Biotec, San Diego, CA), CD24-PerCP-Cy 5.5 (BD Biosciences), CD15-V450 (BD Biosciences), and ALDHA1-FITC (USBiological, Salem, MA). Cell viability was examined using far red fluorescent reactive dye (Thermo Fisher, Waltham, MA). Cells were incubated with the antibodies for 30 min at 4°C, acquired on a FACSCalibur flow cytometer or FACSVerse (Becton Dickinson, Franklin Lakes, NJ), and analyzed using FlowJo software (TreeStar, Inc., Ashland, OR). Isotype control staining was < 5% for all samples analyzed.
Cd24 percp cy5
Manufactured by BD
Sourced in United States
CD24-PerCP-Cy5.5 is a fluorescent-conjugated antibody used for the detection and analysis of CD24 expression on cell surfaces using flow cytometry. It consists of an anti-CD24 monoclonal antibody coupled with the PerCP-Cy5.5 fluorescent dye.
Automatically generated - may contain errors
Lab products found in correlation
Cd44 apc, by BD (2 mentions)
Cd44 fitc, by BD (2 mentions)
Facsverse, by BD (2 mentions)
Facscalibur flow cytometer, by BD (2 mentions)
Hla abc fitc, by BD (1 mentions)
Cd15 v450, by BD (1 mentions)
Ifn γ, by R&D Systems (1 mentions)
Cd133 pe, by Miltenyi Biotec (1 mentions)
Ionomycin, by InvivoGen (1 mentions)
Fix perm kit, by BD (1 mentions)
Live dead fixable aqua dead cell stain kit, by Thermo Fisher Scientific (1 mentions)
Cd4 pacific blue, by BD (1 mentions)
Lrsii, by BD (1 mentions)
Cd10 pe cy7, by BD (1 mentions)
Phorbol myristate acetate (pma), by InvivoGen (1 mentions)
Cd71 fitc, by BD (1 mentions)
Cd25 pe, by BD (1 mentions)
Brefeldin a, by BD (1 mentions)
Cd38 pe, by BD (1 mentions)
Anti rabbit igg, by Cell Signaling Technology (1 mentions)
12 protocols using cd24 percp cy5
1
Chordoma Cell Phenotype Changes by IFN-γ
2
Characterization of PBMC Immune Profiles
3
Comprehensive Antibody Characterization for EMT Research
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Zyxin antibody was from Santa Cruz (sc‐293448) at 1:250 dilution for WB, 1:100 dilution for IF). The following antibodies were from Cell Signaling Technology: ZEB1 (3396S, 1:1000 dilution for WB), GAPDH (2118S, 1:5000 dilution for WB), E‐Cadherin (14472S, 1:1000 dilution for WB, 1:100 dilution for IF), vimentin (5741S, 1:1000 dilution for WB, 1:100 dilution for IF), CD44 (3570S, 1:300 dilution for immunohistochemistry [IHC]), 1:400 dilution for IF), SIRT1 (8469S, 1:1000 dilution for WB, 1:100 dilution for IP), 1:100 dilution for IF), HA‐Tag (3724S, 1:50 dilution for IP), anti‐mouse IgG (HRP‐linked, 7076S, 1:5000 dilution for WB), anti‐rabbit IgG (HRP‐linked, 7074S, 1:5000 dilution for WB). CD44‐PE (550989, 1:50 dilution for FCM) and CD24‐PerCP‐Cy5.5 (561647, 1:50 dilution for FCM) were from BD; CD24 (ab202073, 1:100 dilution for IF), H3 K9 (ab4441, 1:10000 dilution for WB), H3 K14 (ab82501, 1 mg/mL for WB), H3 K23 antibody (ab177275, 1:1000 dilution for WB), H3 K27 Antibody (ab4729, 1 μg/mL for WB), and H3 (ab47915, 1:1000 for WB) were from Abcam. Alexa Fluor 488 goat anti‐mouse IgG1 (A21121, 1:500 dilution for IF), Alexa Fluor 488 goat anti‐mouse IgG(H+L) (A11001, 1:500 dilution for IF), Alexa Fluor 568 goat anti‐rabbit IgG(H+L) (A11010, 1:500 dilution for IF), and Alexa Fluor 568 goat anti‐mouse IgG2a (A21134, 1:500 dilution for IF) were from Thermo Fisher Scientific.
4
Immunophenotyping of B cell subsets in severe asthma
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Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Paque (GE Healthcare, Marolles-en-Hurepoix, France) gradient centrifugation and frozen. Immunophenotyping of PBMCs from 9 severe asthmatic patients was performed using flow cytometry. PBMCs from 10 HV were analyzed as controls. PBMCs were rapidly thawed by placing cryovials at 37°C, washed and stained according to standard protocols using the following mAbs: CD19-BUV395, CD27-BUV737, CD38-BV605, CD24-PerCP-Cy5.5, and CD9-BV510 (BD Biosciences, Le Pont de Claix, France). These markers were used to distinguish CD19+ B lymphocytes, CD19+CD27+ memory cells, CD19+CD27− naïve cells, CD19+CD24hiCD38hi transitional cells, CD19+CD24−CD38+ plasma cells, and CD19+CD9+ Bregs. For all experiments, dead cells were excluded using the Zombie NIR™ Fixable Viability kit (BioLegend, London, UK). Human anti–IL-10 (BD Biosciences) was used to inhibit the IL-10 pathway. Samples were assessed on a BD LSRFORTESSA X-20 (BD Biosciences, Le Pont de Claix, France), and the data were analyzed using FlowJo v10 software (FlowJo LLC, Ashland, OR, USA).
5
Isolation and Characterization of PBMCs
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Peripheral blood mononuclear cells (PBMCs) were obtained from healthy individuals homozygous for the FcγRIIB-T232 or FcγRIIB-I232 site, under appropriate ethics approval from the NIHR Cambridge Bioresource. Inclusion criteria for individuals were people aged between 44 and 77 years, with no serious co-morbidities, no direct family history of autoimmune disease, no use of immunosuppressants or steroids and no hospitalisation within the last 12 months. Individuals were age and sex matched (18 females and 11 males matched between genotypes). Flow sorting was performed using CD19-BV785, CD38-BV711, CD3-NC650, CD14-605NC, CD24-PerCP-Cy5.5, IgD-FITC, CD27-PE-Cy7 (all from BD Bioscience) and Aqua (for live-dead cell detection, Invitrogen), where flow protocol is outlined in Supplementary Fig. 7 .
6
Quantifying CD44+/CD24- Expression under Hypoxia
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Single cell suspensions for flow cytometry were achieved by passing the cells through a 40 μm cell strainer (BD Falcon, BD Biosciences, Franklin Lakes, NJ, USA) and staining with CD44-APC (#559942) and CD24-PerCP-Cy5.5 (#561647) from BD Pharmingen in Hanks’ buffer supplemented with 2% fetal bovine serum (FBS) according to the manufacturer’s instructions. All stained cells were run in a BD FACS Canto II (BD Biosciences, San Jose, CA, USA), and data were analyzed using FCS Express 5.0 software (De Novo Software, Glendale, CA, USA). To assess the changes in the expression of CD44+/CD24− under hypoxia, as compared to normoxia, gates were first established for positivity stained normoxic cells with antibodies of CD44 and CD24 using unstained normoxic cells as a negative control. Then, the stained normoxic group was chosen as a control and the same gating was adopted to measure the expression of CD44+/CD24− in hypoxic cells or other treatments.
7
Comprehensive B-Cell and cTfh Immunophenotyping
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B‐cell subsets and cTfh were characterized using following surface markers: CD19‐FITC (Fluorescein isothiocyanate; Clone J4.119, Beckman Coulter, Indianapolis, IN, USA), CD38‐APC (Allophycocyanin; Clone HB‐7, BD Biosciences, San Jose, CA, USA), CD24‐PerCP‐Cy5.5 (Peridinin chlorophyll protein complex‐cyanine 5.5; Clone ML5, BD Biosciences), CD27‐PE (phycoerythrin; Clone L128, BD Biosciences), CD4‐PC7 (PE‐Cyanine7; Clone 13B8.2, Beckman Coulter), CXCR5‐ Alexa Fluor®488 (Clone RF8B2, BD Biosciences), PD‐1‐PerCP‐Cy5.5 (Clone EH12.1, BD Biosciences), and ICOS‐APC (Clone ISA‐3, BD Biosciences). The gating strategy of B‐cell subsets is shown in Supplementary Figure 1 , while that of cTfh is shown in Supplementary Figure 2 .
8
Flow Cytometry Analysis of Irradiated Tumor Cells
9
CD24 and CD44 Expression Analysis in Morphine-Treated Cells
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To evaluate CD24 and CD44 expression, cells were cultured with morphine (10 μM) for 14 days. Cells (2 × 106) were harvested and incubated with antibodies against CD44 FITC (BD Pharmingen) and CD24 PerCP-Cy5.5 (BD Pharmingen) for 30 minutes at 4°C in the dark. Unbound antibody was washed away through two cycles of washing with PBS. Then cells were analyzed on a BD FACS Calibur flow cytometer. These data were analyzed by Cellquest Pro and at least 20,000 events per sample were collected.
10
Single Cell Isolation for FACS Analysis
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Single cell suspensions for FACS and flow cytometry are achieved by passing cells through 40 μm cell stainer (BD Falcon) and staining with CD44-APC (#559942) and CD24-PerCP-Cy5.5 (#561647) from BD Pharmingen in Hanks' buffer supplemented with 2% FBS. Cells were collected in Hanks' buffer supplemented with 50% FBS.
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