Flow cytometry was performed using a FACScan instrument (BD Biosciences, San Jose, CA, USA). NDY-1 cells were dissociated and washed once in phosphate buffered saline (PBS) containing 1-2% bovine serum albumin and 5 mM EDTA; they were subsequently stained with anti-CD24-FITC (Invitrogen) and anti-CD44-PE (Invitrogen) at a concentration of 10 μl of antibody per 106 cells before being incubated at 4°C for 15-30 min. Cells were washed once with PBS buffer, and flow cytometry was performed to detect CD44+CD24- cells using each fluorescence channel. Gates were determined by analyzing the unstained cells and single stains. ALDEFLUOR fluorescence was detected using the green fluorescence channel (530 ± 15). Data for 10,000 cells were collected and analyzed using the Cell Quest software version 3.3 (BD Biosciences).
Anti cd24 fitc
Manufactured by Thermo Fisher Scientific
Sourced in United States
Anti-CD24-FITC is a fluorescently-labeled monoclonal antibody that binds to the CD24 cell surface antigen. It is commonly used in flow cytometry applications to identify and quantify CD24-expressing cells.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd44 pe, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Cellquest software version 3, by BD (1 mentions)
Facscan, by BD (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Facsaria, by BD (1 mentions)
Anti cd4 fitc, by Thermo Fisher Scientific (1 mentions)
Anti ifn γ pe, by Thermo Fisher Scientific (1 mentions)
Anti il 10 pe, by Thermo Fisher Scientific (1 mentions)
Anti cd3 pe, by BD (1 mentions)
Anti cd24 fitc, by BD (1 mentions)
Anti cd38 pe, by BD (1 mentions)
Anti tnf α fitc, by BD (1 mentions)
Anti il 17a apc, by Thermo Fisher Scientific (1 mentions)
Anti cd19 percp cy5, by Thermo Fisher Scientific (1 mentions)
Anti cd38 pe, by Thermo Fisher Scientific (1 mentions)
Anti il 6 apc, by BD (1 mentions)
Anti cd24 alexa fluor647, by BD (1 mentions)
Permeabilization and ic fixation buffers, by Thermo Fisher Scientific (1 mentions)
Coumarin 6, by Merck Group (1 mentions)
9 protocols using anti cd24 fitc
1
Flow Cytometric Characterization of CD44+CD24- Cells
2
Isolation and culture of glioma stem cells
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Gentle MACS dissociators were utilized to dissociate and homogenize the glioma tissue into primary glioma tissue cells (GTCs), which were cultured in multipotent adult stem cell medium (60% DMEM low glucose, 40% MCDB-201, 2% fetal bovine serum, 10 ng/ml hPDGF, 10 ng/ml hEGF, 1 mg/ml linoleic acid–BSA, 10−9 M dexamethasone, 1 × ITS, and 10−4 M ascorbic acid-2 phosphate). After reaching 80% confluence, the GTCs were detached with trypsin and then stained with anti-CD24-FITC and anti-CD44-PE (Invitrogen, Carlsbad, CA, USA) antibodies (10 μg per 106 cells). The CD44+CD24− cells were considered to be GSCs (10 (link)). Microglia cells (HMC3, ATCC® CRL-3304™) were obtained from the American Type Culture Collection (ATCC, VA, USA), which were stimulated with lipopolysaccharide (LPS, 1 μg/ml) and cultured in RPMI-1640 medium (Gibco, Scoresby, VIC, Australia) supplemented with 10% FBS and 1% glutamine. All the cells were maintained in a humidified incubator (37°C, 5% CO2).
3
Isolation and Characterization of Glioma Stem Cells
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Glioma cells were cultured in Dulbecco's modified Eagle medium (DMEM)/high glucose with 10% fetal bovine serum (FBS, Gibco, Carlsbad, CA, USA) and were maintained in a humidified incubator at 37°C with 5% CO2. GSCs were separated as described previously from tissue cells.23 , 24 Primary cells were detached with trypsin, washed once in FACs buffer (PBS containing 1%‐2% BSA and 5 mM EDTA), then stained with anti‐CD24‐FITC (Invitrogen, Carlsbad, CA, USA) and anti‐CD133‐PE (Invitrogen) using 10 μl of antibody per 106 cells, and incubated at 4°C for 15 minutes. Following incubation, cells were washed once with FACs buffer. The CD44+CD24− cells were considered to be GSCs.
4
Multiparametric Flow Cytometry of Immune Cells
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Flow cytometry was performed with the following antibodies: anti-CD19-Percp-Cy5.5, anti-CD19-Percp-eFluor710, anti-CD24-FITC, anti-CD38-superbright600, anti-CD38-PE, anti-Ki-67-Alexa Fluor647, anti-IL-10-PE, anti-TIM-1-PE, anti-CD4-FITC, anti-IFN-γ-PE, anti-IL-17A-APC (eBioscience, San Diego CA, USA), anti-IL-6-APC, anti-TNF-α-FITC, anti-IL-12-BV-421, and anti-CD24-Alexa Fluor647 (BD Biosciences, France). Intracellular cytokines and Ki-67 were assessed in cells treated with Permeabilization and IC Fixation Buffers (eBioscience, San Diego, CA, USA). Stained cells were analyzed with a 21-color ZE5 cell analyzer (Bio-Rad, USA). The CD19+CD24hiCD38hi B subset was sorted (FACSAria; BD Pharmingen) using anti-CD19-Percp-Cy5.5, anti-CD24-FITC, and anti-CD38-PE. The sort purity of CD19+CD24hiCD38hi B cells was routinely >90%. CD4+T subpopulation was sorted (FACSAria; BD Pharmingen) using anti-CD38-PE and anti-CD3-PE. The sort purity of CD4+T cells was routinely >95%. All flow cytometry data were analyzed with FlowjoV10 Software (Tree Star, OR, USA).
5
Comprehensive Nanomedicine Protocol Synthesis
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Arsenic trioxide, Igepal CO-720, coumarin 6 and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma‒Aldrich (St. Louis, MO, USA). Manganous acetate and chitosan were obtained from Macklin Biochemical Co., Ltd. (Shanghai, China). MPEG5K-PLGA20K and mPEG5K-PLA20K were purchased from Yare Biotech, Inc. (Shanghai, China). Mal-PEG5K-PLGA20K was synthesized in Hunan HuaTeng Co., Ltd. (Changsha, China). DSS6 (Ac-CDSSDSSDSSDSSDSSDSS) peptide was synthesized in Top-peptide Co., Ltd. (Shanghai, China). Hydroxyapatite (HA) was obtained from Emperor Nano Material Co., Ltd. (Nanjing, China). 1,1ʹ-Dioctadecyl-3,3,3ʹ,3ʹ-tetramethylindotricarbocyanine iodide (DiR) was purchased from Meilun Biotech Co., Ltd. (Dalian, China). CellTracker Green 5-chloromethylfluorescein diacetate (CMFDA) was purchased from Yeasen Biotech Co., Ltd. (Shanghai, China). Puromycin dihydrochloride was purchased from Beyotime Biotech Co., Ltd. (Shanghai, China). Anti-CD44-APC and anti-CD24-FITC were purchased from eBioscience (San Diego, CA, USA). MethoCult™ SF H4636 was purchased from STEMCELL Technologies Inc. (Vancouver, Canada).
6
Flow Cytometric Analysis of Cell Markers
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The following antibodies were used for flow cytometric analysis of cells: anti-CD24-FITC, anti-CD44-FITC, anti-CD90-FITC, anti-CD133-PE, anti-MDR1-PE, anti-EpCAM-PE, anti-CD13-PE (all from eBioscience, San Diego, CA) and anti-CDCP1-FITC and anti-ABCG2-PE (both from BioLegend, San Diego, CA). Cells were incubated with the indicated antibodies for 60 min and were then washed twice with PBS containing 2% FCS. Flow cytometric analysis was performed using a FACS-Calibur (BD Immunocytometry Systems, Franklin Lakes, NJ).
7
Phenotyping of B-cell Subsets in PBMCs
8
Phenotypic Characterization of B Cell Subsets
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Peripheral venous whole blood was collected from subjects, and 0.83% ammonium
chloride was used to separate red blood cells. Whole blood was then stained with
the following fluorescent antibodies: anti-CD24-FITC, anti-CD19-PE,
anti-CD27-PEcy5, and anti-CD38-APC (eBioscience, San Diego, CA, USA).
Fluorescently-stained cells were then detected by BD FACSVerse flow cytometry
(BD Biosciences, San Jose, CA, USA). Peripheral venous blood B cells
(CD19+ lymphocytes) were divided into Breg cells
(CD19+CD24hiCD38hi), memory B cells
(CD19+CD27+CD24hi), and plasma cells
(CD19+CD27hiCD38hi).
chloride was used to separate red blood cells. Whole blood was then stained with
the following fluorescent antibodies: anti-CD24-FITC, anti-CD19-PE,
anti-CD27-PEcy5, and anti-CD38-APC (eBioscience, San Diego, CA, USA).
Fluorescently-stained cells were then detected by BD FACSVerse flow cytometry
(BD Biosciences, San Jose, CA, USA). Peripheral venous blood B cells
(CD19+ lymphocytes) were divided into Breg cells
(CD19+CD24hiCD38hi), memory B cells
(CD19+CD27+CD24hi), and plasma cells
(CD19+CD27hiCD38hi).
9
Cell Surface Marker Detection and Isolation
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For surface marker detection, cells were resuspended in 100 μL Hank’s balanced salt solution with 1% FBS (Gibco). For isolation of CD133+ cells for western blot analysis, cells were resuspended in 100 μL Hank’s balanced salt solution with 1% FBS. Fc Receptor Binding Inhibitor (eBioscience, Inc., San Diego, CA, USA) was added and the sample was incubated for 5 min at 4 °C. After two washes, Anti-CD133 fluorescein isothiocyanate (FITC) (Biorbyt, Cambridge, UK), Anti-CD24 FITC (eBioscience), Anti-CD44 PE-Cy5 (eBioscience) or Anti-ESA PE (eBioscience) were added and the sample was incubated for 30 min at 4 °C. After two washes, the proportions of subpopulation cells that expressed the different surface markers were determined using a FACSCalibur system (BD Biosciences, San Jose, CA, USA) and cell sorting of CD133+ cells was done using a FACSAria system (BD Biosciences). Side scatter and forward scatter profiles were used to eliminate cell doublets.
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