We collected 2.5 ml of peripheral blood samples from ESCC patients and healthy volunteers in EDTA tubes. Density gradient centrifugation was performed using the RosetteSep™ Human Circulating Epithelial Tumor Cell Enrichment Cocktail (StemCell™ Technologies, Vancouver, Canada) combined with Lymphoprep™ (StemCell™ Technologies, Vancouver, Canada). To the 2.5 ml blood sample was added 250 µl (50 µl/ml) of the RosetteSep™ cocktail and then incubated for 20 min at room temperature. Blood samples were diluted with equal volumes of PBS and carefully layered onto Lymphoprep™ then centrifuged at 3,600 rpm at room temperature for 20 min. After centrifugation, supernatants were transferred to another 15 ml conical tube with cells pelleted by centrifugation at 1,800 rpm for 20 min at room temperature. The enriched cells were collected, red blood cells were lysed by BD Pharm Lyse lysing solution (Becton, Dickinson and Company, New Jersey, USA), and washed in PBS.
Rosettesep human circulating epithelial tumor cell enrichment cocktail
Manufactured by STEMCELL
Sourced in Canada
RosetteSep™ Human Circulating Epithelial Tumor Cell Enrichment Cocktail is a laboratory reagent used to enrich circulating epithelial tumor cells from human whole blood samples. It functions by cross-linking unwanted cells to red blood cells, allowing the desired cells to be easily separated.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 594, by Thermo Fisher Scientific (2 mentions)
Anti cd44, by Cell Signaling Technology (2 mentions)
Rosettesep human cd45 depletion cocktail, by STEMCELL (2 mentions)
Anti ck19, by Abcam (2 mentions)
Anti epcam, by Abcam (2 mentions)
Anti dclk1, by Abcam (2 mentions)
Anti anxa2, by BD (2 mentions)
Anti cd45, by Abcam (2 mentions)
Anti gpcr gpr49 lgr5, by Abcam (2 mentions)
Alexa fluor 488 coupled secondary igg, by Thermo Fisher Scientific (2 mentions)
Anti β actin total, by Merck Group (2 mentions)
Easysep human whole blood cd45 depletion kit, by STEMCELL (2 mentions)
Pharm lyse lysing solution, by BD (1 mentions)
Lymphoprep, by STEMCELL (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
N2 supplement, by Thermo Fisher Scientific (1 mentions)
24 well ultra low attachment plate, by Corning (1 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (1 mentions)
5 protocols using rosettesep human circulating epithelial tumor cell enrichment cocktail
1
Enrichment of Circulating Epithelial Tumor Cells
2
Isolation and Characterization of Circulating Tumor Cells
3
Isolation and Characterization of Circulating Tumor Cells
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Antibodies used include: anti-CD44 (Cell Signaling Technology, Danvers, MA); anti-DCLK1, anti-CD45, anti-EpCAM, anti-CK19 and anti-GPCR GPR49 (Lgr5) (Abcam, Cambridge, MA); anti-AnxA2 (BD Biosciences, Carlsbad, CA), and anti-β-actin (total) (Sigma, St Louis, MO). Anti-PG antibody was generated in our laboratory as described [27 (link)). Alexa Fluor-594 and Alexa Fluor-488 coupled secondary IgG were from Invitrogen (Carlsbad, CA). Three kits from Stem Cell Technologies were used: 1) RosetteSep™ Human CD45 Depletion Cocktail (#1522), 2) RosetteSep™ Human Circulating Epithelial Tumor Cell Enrichment Cocktail (#15127), and 3) EasySep™ Human Whole Blood CD45 Depletion Kit (#18289), (Vancouver, Canada).
4
Circulating Tumor Cell Culture Protocol
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The blood collected in EDTA tubes was immediately used for CTC culture. First, viable CTCs were enriched through depletion of hematopoietic CD45(+) cells using the RosetteSep Human Circulating Epithelial Tumor Cell Enrichment Cocktail (STEMCELL TECHNOLOGIES, Vancouver, Canada), according to the manufacturer’s protocol. Then, CD45(−) CTCs were incubated at 37 °C in 24-well non-adherent plates with CTC culture medium (RPMI1640 with EGF and FGF-2, Insulin-Transferrin-Selenium supplement, L-Glutamine), as previously described13 (link). Colospheres and tumor cells were observed during the first weeks of culture and were then transferred to new 24-well plates for further growth and to T25 flasks for cell expansion. In these conditions, CTCs could be quickly expanded and after a few months, billions of tumor cells were obtained.
5
Enrichment and Culture of Circulating Tumor Cells
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On the day of surgery, hospital shipped two EDTA tubes of blood for each patient through a taxi which was dedicated to this project and during the whole project, blood had never waited more than 4 hours following the sampling to be processed, the average time being 2 hours. Blood samples were then pooled to reach a total volume of 8–10 mL, they were then incubated at room temperature for 20 min with 50 µL of Rosette Sep Human Circulating Epithelial Tumor Cell Enrichment Cocktail (STEMCELL Technologies) per mL of blood diluted in phosphate buffered saline (PBS) containing 2% of fetal bovine serum (FBS) v/v. After 20 min, the mix was gently put on 15 mL of lymphocyte separation medium (LSM, Eurobio) and centrifuged 20 min at 1200g without brake. Cells located at the interface between serum and LSM were delicately harvested, washed twice in PBS containing 2% of FBS and resuspended in M12 medium (1 mL/well) in ultralow attachment 24-well plates (Corning). M12 medium contains advanced DMEM-F12 (Gibco), 2 mmol/L of l -glutamine, 100 Unit/mL of penicillin and streptomycin, N2 supplement (Gibco), 20 ng/mL of epidermal growth factor (R&D) and 10 ng/mL of fibroblast growth factor-basic (R&D). CTC41 has been partially described previously.64 (link)
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