The small intestine crypts were isolated at day 5 post-irradiation. The crypts were incubated in the digestive juice, which pre-warmed and contained 2 mL TRYPLE express (Gibco Life Technologies), 20 µL Y27632 (Sigma-Aldrich), 2 mL N-acetylcysteine (Sigma-Aldrich), 80 µL DNase and 2 µL Dispase Type II (Gibco Life Technologies) in 37 °C for 8 minutes. Quickly, they ended the digestive process by adding DMEM with 10% fetal bovine serum (Gibco life technology). The mixture was centrifuged at 1,200 rpm for 5 min at 4 degrees. The cells were passed through 40-µm strainers (BD Bioscience) and resuspended with ABS (~1×106 cells/100 µL). The cells were counterstained with different fluorescein antibodies, including APC anti-mouse/human CD44 (Biolegend), PE anti-mouse CD45 (Biolegend), FITC anti-mouseCD326 (Biolegend) and propidium iodide (PI, Sigma-Aldrich), according to the manufacturer’s instructions. PI(−) CD45(−) EpCAM(+) CD44(+) [marked as CD44(+)] cells are sorted using s flow cytometer (Beckman). The sorted cells were collected, and their RNA was extracted using the RNeasy Plus Mini Kit (Qiagen). The GENEWIZ company did the RNA-seq and analysis work.
Dispase type 2
Manufactured by Thermo Fisher Scientific
Sourced in United States
Dispase type II is a neutral protease derived from Bacillus polymyxa. It is commonly used for the gentle dissociation of a variety of cell types, including epithelial, endothelial, and primary cells, from their substrates.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Tissue culture plates, by Eppendorf (2 mentions)
Collagenase type 9, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Pe anti mouse cd45, by BioLegend (1 mentions)
Apc anti mouse human cd44, by BioLegend (1 mentions)
Dnase, by Thermo Fisher Scientific (1 mentions)
Tryple express, by Thermo Fisher Scientific (1 mentions)
Y 27632, by Merck Group (1 mentions)
N acetylcysteine, by Merck Group (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Rneasy plus mini kit, by Qiagen (1 mentions)
Leibovitz l 15 media, by Thermo Fisher Scientific (1 mentions)
Collagenase type 2, by Thermo Fisher Scientific (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Collagenase type 4, by Thermo Fisher Scientific (1 mentions)
Matrigel, by Corning (1 mentions)
Collagenase type 2, by Merck Group (1 mentions)
6 protocols using dispase type 2
1
Isolation and Characterization of Intestinal Crypt Stem Cells
2
Isolation and Dissociation of Trigeminal Ganglia
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Bilateral TGs were isolated by removing the skull cap and brain to expose the TGs readily visible in the base of the cranium. Using spring-loaded scissors the TGs were dissected and placed into cold HBSS and dissociated based on the previously described protocol51 (link). Briefly, TG were minced and incubated in HBSS containing collagenase type II (Gibco) and dispase type II (Gibco) at 37 °C for 20 min and mechanically dissociated using a fire polished glass Pasteur pipette. A 12.5% by 28% percoll gradient was used to separate myelin and nerve debris from TG neurons. Cells were then plated in DMEM/F12 (Gibco) containing 5% fetal bovine serum and antibiotics (penicillin/streptomycin, 50 U/mL) on 5 mm coverslips. Coverslips were flooded 2 h later with Leibovitz L-15 media (Gibco) containing 10% fetal bovine serum, 5 mM HEPES and 5 mM glucose, and used at room temperature. Experiments were performed within 8 h of tissue harvest.
3
Isolation of Colon Cancer Cells from Surgical Specimens
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Surgically resected colon cancer tissues were obtained from patients from Samsung Medical Center (SMC), Seoul, Korea. This study was approved by the Institutional Review Board (IRB) of SMC (IRB No. 20**-0*-017). Tissues were washed three times with 70% ethanol, followed by cold PBS washes until the supernatant was clear. Next, the tissues were chopped into ~5-mm pieces and further washed with cold PBS. These pieces were then incubated with digestion buffer (Dulbecco’s modified Eagle medium with 2.5% FBS, 1% penicillin/streptomycin (Invitrogen, Carlsbad), 75 U/mL collagenase type IV (Gibco, Grand Island, NY), and 125 µg/mL dispase type II (Gibco, Grand Island, NY)) at 37 °C for 30–60 min. Digested cells were then centrifuged at 200 g for 3 min to separate adenoma from single cells. Dissociated cells were passed through a 40-µm cell strainer and washed several times with PBS. Isolated colon cancer cells were counted and embedded in Matrigel on ice and seeded into 24-well cell culture plates.
4
Isolation and culture of colon cancer cells
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The Ethics Committee of the Medical Research Council of Hungary (ETT-TUKEB, No. 51323-4/2015/EKU) approved all experiments with human samples and informed consent was obtained from the patients. Samples were collected at the Department of Oncosurgery, Uzsoki Hospital, Budapest, Hungary. Patient data are shown in Supplementary Tables S1 , S2 . Tumor and normal colon tissue dissected at a distance >5 cm from the tumor were isolated and cut into small pieces (<0.5 cm) in PBS. After extensive washing with PBS three times, tissue pieces were incubated in a digestive mix [DMEM high glucose with 20% FBS, 75 U/mL collagenase type IX (Sigma), and 125 μg/mL dispase type II (Invitrogen, Carlsbad, CA, United States)] for 2 h at 37°C with extensive shaking. After removal of tissue pieces, single cells were then centrifuged at 300 g for 5 min, washed twice in PBS and cultured in tissue culture plates (Eppendorf) in fibroblast medium containing 15% FBS.
5
Isolation of Metastatic CRC Tumor Cells
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Tumor samples from metastatic CRC patients were collected under a Duke IRB approved protocol (Pro00002435) at Duke University Hospital. All participants provided written informed consent to participate in the study. Tumor samples were chopped into 5 mm pieces and washed with PBS several times. The tumor fragments were incubated in digestion buffer (Dulbecco's modified Eagle medium with 2.5% fetal bovine serum, penicillin/streptomycin [Invitrogen], 75 U/mL collagenase type IX [Sigma], 125 μg/mL dispase type II [Invitrogen]) for 60 min at 37 °C. The supernatant was collected in a 50 mL Falcon tube, centrifuged at 1000RPM for 5 min, and then washed with PBS repeatedly. Isolated cancer cells were counted using a hemocytometer. Single cells were embedded in ice cold Matrigel (Corning Life Sciences) and seeded in 24-well plates. Matrigel was polymerized for 10 min at 37 °C. Basal culture medium was supplemented with a combination of growth factors as previously described.13 (link)
6
Isolation and culture of tumor cells
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The Ethics Committee of the Medical Research Council of Hungary (ETT-TUKEB, No. 51323-4/2015/EKU) approved all experiments with human samples and informed consent was obtained from patients. Samples were collected at the Department of Oncosurgery, Uzsoki Hospital, Budapest, Hungary. Tumor tissues were cut into small pieces (<0.5 cm), and after washing with PBS three times, tissue pieces were incubated in a digestive mix (DMEM high glucose with 20% FBS, 125 µg/mL dispase type II (Invitrogen), 75 U/mL collagenase type II (Merck), 1 h) at 37 °C with shaking. Samples were then centrifuged at 300× g for 5 min to isolate single cells, they were washed twice in PBS and cultured in tissue culture plates (Eppendorf, Vienna, Austria) in DMEM high glucose, 10% FBS, penicillin/streptomycin and glutamine (fibroblast medium). In some experiments 5 nM factor VIIa, 10 ng/mL TGFβ, or 5 ng/mL IL1α were used for 4 days in serum-free medium. Patient data are shown in Table S2 .
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