1×106/ml HCC cells were collected in 100ul PBS. Anti-EPCAM (eBioscience, USA) and Anti-CD133 (eBioscience, USA) were added into HCC cells suspension and incubated for 30 minutes on ice. Then wash the cell pellet with PBS. Resuspend cell pellet with fresh PBS and analysis with flow cytometry.
Anti cd133
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
Anti-CD133 is a laboratory reagent used for the detection and analysis of CD133, a cell surface glycoprotein. It is commonly used in flow cytometry and cell sorting applications to identify and isolate CD133-positive cells. The product is intended for research use only and its specific applications and performance characteristics may vary depending on the user's protocols and instrumentation.
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Lab products found in correlation
Anti epcam, by Thermo Fisher Scientific (2 mentions)
Anti ahr, by Abcam (2 mentions)
Moflo xdp, by Beckman Coulter (1 mentions)
Anti cd44, by Thermo Fisher Scientific (1 mentions)
Anti nanog, by Thermo Fisher Scientific (1 mentions)
Anti human cd44, by Thermo Fisher Scientific (1 mentions)
Accutase, by Merck Group (1 mentions)
Facscelesta, by BD (1 mentions)
Aldefluor assay, by STEMCELL (1 mentions)
Confocal laser scanning microscope, by Leica camera (1 mentions)
Phospho stat1, by Abcam (1 mentions)
Alexa fluor 488 conjugated chicken anti mouse, by Thermo Fisher Scientific (1 mentions)
Vectashield mounting media, by Vector Laboratories (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
Doublecortin, by Abcam (1 mentions)
Fluorescent microscope, by Zeiss (1 mentions)
Anti brdu, by BD (1 mentions)
Anti gfap, by Merck Group (1 mentions)
Cy2 or cy3 conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Superfrost plus slide, by Thermo Fisher Scientific (1 mentions)
13 protocols using anti cd133
1
Immunophenotyping of HCC Cells
2
Isolation and Culture of Prostate Cancer Stem Cells
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Human prostate cancer DU145 cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The DU145 cells were grown in Roswell Park Memorial Institute-1640 medium. The medium was supplemented with 10% fetal bovine serum, 4 mL glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin. The cells were incubated at 37°C in a humidified atmosphere of 5% CO2.
The DU145 cells were counted and resuspended in the flow buffer (1 × 107 cells were resuspended in 500 µL of buffer). Flow buffer: PBS, pH = 7.2, 0.5% BSA, and 2 mol ethylenediaminetetraacetic acid were added. The cells were incubated with anti-CD133 (eBioscience, California, USA), anti-CD44 (eBioscience), and anti-NANOG (Invitrogen, California, USA) antibodies at 4°C for 30 min in the dark. The cells were then resuspended in 500 µL flow buffer. The CD44+/CD133+/NANOG+ PCSCs were sorted by flow cytometry (Beckman MoFloXDP). PCSCs were cultured in a complete growth medium supplemented with 10% serum.
The DU145 cells were counted and resuspended in the flow buffer (1 × 107 cells were resuspended in 500 µL of buffer). Flow buffer: PBS, pH = 7.2, 0.5% BSA, and 2 mol ethylenediaminetetraacetic acid were added. The cells were incubated with anti-CD133 (eBioscience, California, USA), anti-CD44 (eBioscience), and anti-NANOG (Invitrogen, California, USA) antibodies at 4°C for 30 min in the dark. The cells were then resuspended in 500 µL flow buffer. The CD44+/CD133+/NANOG+ PCSCs were sorted by flow cytometry (Beckman MoFloXDP). PCSCs were cultured in a complete growth medium supplemented with 10% serum.
3
Characterizing Cancer Stem Cells with Flow Cytometry
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Cells were harvested with Accutase (Sigma-Aldrich) and stained either with the Aldefluor assay (Stem cell technologies) according to the manufacture’s recommendation or in PBS buffer with 5% FBS, 1% HEPES and 0.5 mM EDTA with anti-human CD44 (1:100, clone: IM7, #48-0441-80, Thermo Fisher) and anti-CD133 (1:50, clone: TMP4, #130133186, eBioscience). To exclude dead cells from the analysis, cells were additionally stained with the viability dye 7-Aminoactionomycin D (1:1000, Thermo Fisher). The samples were acquired with the BD FACSCelesta and analyzed with FlowJo software (version 10.9.0). The ALDH+ population was gated based on background fluorescence from the negative control, the diethylaminobenzaldehyde (DEAB) treated cells. The other CSC markers were gated according to the isotype controls (IgG-eFluor 450, eBioscience, #48-403182; rat anti-mouse IgG1-PE, eBioscience, #130-113-200).
4
Immunofluorescence Analysis of Cellular Markers
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Cells were plated on fibronectin (1 μg ml−1)-coated coverslips, fixed in 4% paraformaldehyde and permeabilized with 0.2% Triton X-100. Fixed cells were blocked in 5% BSA and probed with monoclonal anti-AhR, phospho-STAT1, Ki 67, (Abcam, UK), or anti-CD133 (eBioscience) antibody in 5% BSA, followed by Alexa Fluor 488-conjugated chicken anti-mouse (Invitrogen, CA, USA). Cells were mounted with Vectashield mounting media containing DAPI (Vector Laboratories, CA, USA). Confocal microscopy was performed on a Leica laser scanning confocal microscope.
5
Immunohistochemical Analysis of Proliferative Cells
6
Immunofluorescence Staining of CD133 and GFAP
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After 20 minutes of incubation at room temperature with paraformaldehyde at a concentration of 4%, the cells were permeabilized with 0.05 percent Triton X-100 dissolved in Phosphate Buffered Saline (PBS). The cells were treated at 4 degrees Celsius overnight with antiCD133 (1 : 200; eBioscience, USA) and antiGFAP (1 : 200; eBioscience, USA) antibodies after being blocked for one hour with bovine serum albumin dissolved in PBS at a concentration of five percent bovine serum albumin for one hour. After removing any excess antibody, the cells were washed with PBS and then subjected to an incubation with a secondary antibody that was conjugated to PE-labelled goat antimouse IgG (1 : 200; Invitrogen, USA). Then, a fluorescence microscope was used to view the cells after they had been counterstained with DAPI (made by Beyotime in China).
7
Isolation of Coronary Artery Endothelial Cells
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Mice were euthanized by CO2, and the coronary arteries were quickly removed and placed in cold PBS. Each artery was digested with 1 mg/mL collagenase I (No. C9891, Sigma) in PBS for 18 minutes at 37 °C. The suspension was dispersed using an 18 G needle syringe and centrifuged at 1100 rpm for 5 minutes after passing through a 40-μm cell filter. Next, at room temperature, the suspension was carefully removed, and ACK (diethyl 2,5-di (thiophen-2-yl) terephthalate) lysis buffer (No. R7757, Sigma) was added to the cell pellet for 3 minutes, centrifuged at 1100 rpm for 5 minutes, and suspended in 500-μL PBS with 1% fetal bovine serum. The target cells were labeled with anti-CD133 (No. 11-1331-82, Invitrogen) and anti-CD144 (No. 562242, BD Biosciences) and incubated on ice for 20 minutes. The cells were washed twice with PBS+1% fetal bovine serum, and the resuscitated cells were sorted with 500 μL PBS+1% fetal bovine serum (BD Aria III).
8
Immunohistochemical Analysis of Inoculation Sites
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The subcutaneous tissues of inoculation sites in mice were surgically excised, fixed in 37% formalin, embedded in paraffin and sectioned for H&E staining. Immunohistochemistry was performed by using the Vectastain Universal Elite ABC kit (Vector Laboratories, CA, USA) according to the supplied instructions. The sections from paraffin embedded tissues were incubated with anti-S100 β (1:200, Millipore, MA, USA) at 4 °C overnight. After development, tissue sections were moderately counterstained with methyl green. In some experiments, immunohistochemistry was implemented by using Tyramide Signal Amplification Plus Cyanine 3/Fluorecein system (PerkinElmer, CA, USA) according to the supplier's instructions. In brief, the sections were dewaxed, rehydrated, quenched endogenous peroxidase, blocked and incubated with anti-S100 β (1:3,000, Millipore, MA, USA), anti-AhR (1:3,000, Abcam, UK), anti-CD133 (Invitrogen, CA, USA), anti-Ki67 (Abcam, UK) and anti-p-STAT1 (1:3,000, CST, MA) at 4 °C overnight. Slides were then incubated sequentially with two HRP-conjugated secondary antibodies for 1 h at room temperature. After developing in Tyramide Signal Amplification Plus Working Solution for 5 min, the slides were counterstained with DAPI and mounted for confocal analysis.
9
Immunofluorescent Phenotyping of Neural Progenitor Cells
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For phenotyping of NPCs by immunofluorescent staining, spheres were dissociated into single cells using 0.25% trypsin and fixed with 4% PFA in PBS. The cells were permeabilized with PBS containing 0.1% Triton X-100 and incubated with anti-GFP (Abcam Ltd), anti-NESTIN (Millipore, Billerica, MA), anti-TUJ1 (SIGMA-Aldrich), anti-MAP2 (Abcam Ltd), anti-CD133 (Thermo Fisher Scientific), anti-NANOG, anti-GFAP (Agilent Technologies, Santa Clara, CA), anti-CNPase (Abcam Ltd), or anti-PCNA (Abcam Ltd.) antibodies. The cells were subsequently incubated with Alexa 488-anti-rabbit IgG, Alexa 488 or Alexa 594-anti-mouse (Abcam Ltd.), Cy3-conjugated anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA), or Alexa 594-conjugated anti-rat IgG (Abcam Ltd.). Images were obtained by confocal microscopy, and the cells were counted manually in a blinded manner.
10
Cell Cycle Analysis and CD133 Detection
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Following transfection, CAL-27 and SAS cells were trypsinized using a trypsin solution without ethylenediaminetetraacetic acid. The cells were then collected by centrifugation at 1000× g for 5 min, washed with ice-cold PBS, and fixed with 70% cold ethanol for storage at 4 °C for 24 h. After another round of centrifugation and washing with cold PBS, the cells were treated with 25 μL Propidium and 10 μL RNase A (Beyotime, Shanghai, China) at 37 °C in the dark for 30 min. For CD133 detection, cells were obtained and stained with anti-CD133 (17–1338-42, Thermo Fisher Scientific, Waltham, MA, USA) at 4 °C in the dark for 30 min. Cell-cycle analysis was conducted using NovoExpress [Version 1.6.2, Agilent, Santa Clara, CA, USA], and the CD133 was analyzed using CytExpert software [Version 2.4.0.28, Beckman Coulter, Brea, CA, USA].
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