Human pancreatic cancer cell lines, PANC1 and AsPC1 were obtained from Korea Cell Line Bank and maintained in RPMI1640 medium (WELGENE, Daegu, Korea) containing 10% heat-inactivated fetal bovine serum (Life Technologies, Grand Island, NY, USA) and antibiotics (100 U/ml of penicillin and 100 μg/ml streptomycin; Life Technologies). They were incubated at 37°C and 5% CO2. AsPC1 cells were cultured, and stained with anti-CD133 antibody (Miltenyi Biotec, Bergisch Gladbach, Germany) for 15 min on ice with rotation. Followed by washing with a buffer (phosphate buffered saline (PBS) containing 1% bovine serum albumin (BSA) and 0.01% sodium azide) twice, CD133+ AsPC1 cells were isolated by FACSAria (BD Bioscience). Isolated CD133+ AsPC1 cells were incubated in CO2 incubator, and subcultured for tumor xenograft.
Anti cd133 antibody
Manufactured by Miltenyi Biotec
Sourced in Germany
The Anti-CD133 antibody is a laboratory reagent used for the detection and isolation of CD133-positive cells. CD133 is a cell surface marker expressed on various stem and progenitor cell types. The antibody can be used in flow cytometry, immunohistochemistry, and cell separation applications to identify and enrich for CD133-positive cell populations.
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Lab products found in correlation
Alexa 488 conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions)
Aldefluor kit, by STEMCELL (2 mentions)
Facscalibur, by BD (2 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Aspc 1, by Korean Cell Line Bank (1 mentions)
Panc 1, by Korean Cell Line Bank (1 mentions)
Rpmi 1640 medium, by Welgene (1 mentions)
Facsaria, by BD (1 mentions)
Macs anti biotin microbeads, by Miltenyi Biotec (1 mentions)
Pterostilbene, by Merck Group (1 mentions)
Laminin, by Koma Biotech (1 mentions)
Cyclin b1, by Cell Signaling Technology (1 mentions)
Nanog, by Cell Signaling Technology (1 mentions)
Mmp 2, by Cell Signaling Technology (1 mentions)
Stat3, by Cell Signaling Technology (1 mentions)
Phospho stat3, by Cell Signaling Technology (1 mentions)
Mmp 9, by Cell Signaling Technology (1 mentions)
Cyclin e1, by Cell Signaling Technology (1 mentions)
21 protocols using anti cd133 antibody
1
Isolation and Characterization of CD133+ Pancreatic Cancer Cells
2
Isolation and Administration of CD133+ ELCs
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BMNCs were obtained as described above. After centrifugation at 450g at 4 °C for 5 min, the BMNCs were suspended in 80 μl MACS buffer and incubated with 20 μl anti-CD133 antibody (Miltenyi Biotec) for 15 min, followed by incubation with 20 μl of MACS anti-biotin-microBeads (Miltenyi Biotec) in 80 μl MACS buffer. CD133+ ELCs were then obtained by magnetic selection. For ELC therapy, 1 × 106 CD133+ ELCs collected from ROSAmT/mG mice were injected via the tail vein into aged mice every other week.
3
Molecular Mechanisms of Resveratrol and Pterostilbene
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Resveratrol and pterostilbene were purchased from Sigma-Aldrich (Saint Louis, MO, USA) and dissolved in dimethyl sulfoxide (DMSO) at a concentration of 100 mM to prepare a stock solution. Matrigel and gelatin were obtained from BD Biosciences (San Jose, CA, USA). Laminin and the Transwell chamber system were obtained from Koma Biotech (Seoul, Korea) and Corning Costar (Acton, MA, USA), respectively. Antibodies against p21, p53, MMP-2, MMP-9, Bcl-2, Bcl-XL, cleaved caspase-3, cleaved caspase-9, cyclin E1, cyclin B1, STAT3, phospho-STAT3, Sox2, Oct4, Nanog, β-actin, rabbit IgG, and mouse IgG were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-CD133 antibody was obtained from MiltenyiBiotec GmbH (BergischGladbach, Germany).
4
Flow Cytometric Analysis of Cancer Stem Cell Markers
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Log phase growing A2780 and A2780-M cells were collected and washed with PBS. Cells were then incubated with PE- or FITC-conjugated antibodies for 30 minutes at room temperature. Flow cytometry analysis was used to determine the expression of these cell surface markers, and the data was analyzed with Cellquest analysis software (Beckman CXP). Anti-CD34 antibody was from Jingqiao(Cat: Zhongshan Jinqiao, Beijing, China). Anti-CD133 antibody was from Miltenyi Biotec (Cat: 5151214480, Colner, Germany). Anti-CD117 (Cat: 11996-R007-PE) and anti-CD44 (Cat: 12211-MM02-FITC) antibodies were from Sino Biological Inc. (Beijing, China). Anti-CD24 antibody was from Biolegend (Cat:31105-PE, California,USA)
5
Isolation and Culture of CD133+ Cells
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Cells (2 × 107) were detached from tissue culture plates with 5 mM EDTA, centrifuged, and incubated with magnetic microbeads conjugated with anti-CD133 antibody (Miltenyi Biotec, Cambridge, MA). The bead-bound cells (CD133+) and unbound cells (CD133–) were separated in QuadroMACS™ Separation Unit (Miltenyi Biotec, Cambridge, MA). The purity of the isolated CD133+ cells was confirmed by flow cytometric analyses, and by Western blot analyses. The isolated CD133+ cells were cultured in stem cell media as described above.
6
CD133+ Cell Identification in KHOS and U2OS
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After drug treatment, the KHOS and U2OS cells were harvested and resuspended with fresh culture medium at a concentration of 1 × 106 cells/ml. Before CD133+ analysis, the cells were placed on ice for at least 30 min, followed by the treatment with anti-CD133 antibody (catalogue #130-105-225; Miltenyi Biotec, Bergisch Gladbach, Germany) for 1 h. After washing three times with precold PBS, the cells were incubated with an Alexa 488-conjugated secondary antibody (Invitrogen) for 45 min and washed with precold PBS again before analysis by a BD FACS caliber flow cytometer (BD Biosciences).
7
CD133-positive Cell Isolation
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After treatment with SFN and/or cisplatin, cells were detached from the dishes with Trypsin-EDTA (Invitrogen) and suspended at 1×105 cells/mL in PBS supplemented with 2% fetal calf serum. These cells were then incubated with an anti-CD133 antibody (Miltenyi Biotec, Bergisch Gladbach, Germany) in a refrigerator (4°C) for 10 minutes. After washing, CD133-positive cells were detected by using a FACSAria Flow cytometer (BD Biosciences, San Jose, CA).
8
Quantifying CD44 and CD133 Expression
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Cells (5 × 105) were harvested and resuspended in 50 μl of PBS and then incubated with anti‐CD44 antibody (PE, 130–098–210, Miltenyi Biotech) or anti‐CD133 antibody (PE, 130–098–826, Miltenyi Biotech) for 30 minutes at 4°C. After incubation, cells were analyzed by flow cytometry (FACSCalibur, BD Biosciences). Nonspecific mouse IgG antibody was used as isotype control for comparison.
9
Multiparameter Sorting of Stem Cells
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Cultured cells were digested with trypsin and centrifuged at 300g for 10 minutes. The cells were resuspended with MACS buffer (Miltenyi Biotec, Bergisch Gladbach, Germany), FcR blocking reagent was added to the resuspended cells, and the cells were incubated at 4 C for 15 minutes. Anti-CD133 antibody (Miltenyi Biotec) and anti-CXCR4 antibody (Miltenyi Biotec) were added to the cells, and the cells were incubated in the dark at 4 C for 30 minutes. After the cells were washed 3 times with MACS buffer, the cells were analyzed and sorted into 4 subgroups based on combined high/low CD133 expression and high/low CXCR4 expression by flow cytometry on a FACScan (BD Biosciences, Franklin Lakes, NJ).
10
Hematopoietic Cell Immunophenotyping
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Nonadherent hematopoietic cells were harvested every other day. Cell number was determined using a hemocytometer, and viability was determined by Trypan Blue exclusion (Gibco, Grand Island, NJ). Hematopoietic cells were stained according to manufacturer's instructions using the appropriate isotype controls and a panel of fluorescein isothiocyanate (FITC)‐conjugated anti‐CD38, CD33, and CD15; PE‐conjugated anti‐CD133, CD7, and CD14; and PerCP‐conjugated anti‐CD34, CD3, and CD45 antibodies (all from BD Biosciences, San Jose, CA) with the exception of the anti‐CD133 antibody (Miltenyi Biotec, Gladbach, Germany). Stained cells were then run on a FACScaliber (BD, San Jose, CA), and the resultant data were analyzed using Cellquest software.
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