Using a flow cytometer and propidium iodide, we examined the cell cycle. Initially, we used 5 mL of 0.1% collagenase enzyme (Invitrogen, USA) for two hours to separate the tissues of the mammary glands. Next, we used a 50 mL Falcon tube with a 0.7 mm nylon mesh to filter the samples. The tubes were twice cleaned, centrifuged for five minutes at 4500 rpm, and then resuspended in PBS. After that, the cells were preserved in ice-cold ethanol and kept at – 20 °C for a whole day. The cells were resuspended in a propidium iodide (PI) solution containing 100 μl (0.02 mg/mL) PI, 0.1% v/v Triton X-100 in PBS, and 50 μl (0.2 mg/mL) RNase A, then washed twice with PBS and incubated for 30 to 60 min in dark under ambient temperature. The samples were examined using a standardize flow cytometer (Applied Biosystem, USA). We assessed the forward (FS) and side (SS) scatter in order to identify individual cells36 (link).
Collagenase enzyme
Manufactured by Thermo Fisher Scientific
Sourced in United States
Collagenase is an enzyme that breaks down collagen, a structural protein found in the extracellular matrix of various tissues. It is used in laboratory applications that require the dissociation or isolation of cells from collagen-rich tissues.
Automatically generated - may contain errors
5 protocols using collagenase enzyme
1
Cell Cycle Analysis via Flow Cytometry
2
Cell Cycle Analysis of Liver Tissue
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Flow cytometry was used to determine the influence of various treatments on the number of cells in each phase of the cell cycle, and it was carried out as previously described by Abdelwahab et al. (2019) (link) with minor changes. Collagenase enzyme (0.1 percent, Invitrogen, United States) was used to lyse freshly dissected liver tissues, which were subsequently mesh filtered (0.7 mm nylon) and centrifuged (4,000 rpm/5 min). The cells were fixed and stained with propidium iodide before being examined with an Attune flow cytometer (Applied Bio-system, United States). Each cell cycle phase’s number of cells was counted and expressed as a percentage of the total number of cells.
3
Cell Cycle Analysis by Flow Cytometry
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Flow cytometry was applied to detect the effect of different treatments on the number of cells in each phase of the cell cycle and was performed as previously described[ 24 ] with some modifications. Briefly, freshly dissected lung tissues were lysed by collagenase enzyme (0.1%, Invitrogen, USA), then mesh filtered (0.7 mm nylon) and centrifuged (4 000 rpm/5 min). After fixation, the cells were stained with propidium iodide and analyzed by the Attune flow cytometer (Applied Bio-system, USA). The number of cells was counted in each cell cycle phase and was presented as a percentage of the total number of cells.
4
CD133+ Cell Isolation from Tumor Tissue
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CD133 positive (CD133+) cells were isolated from freshly resected and physically/enzymatically dissociated tumor tissue samples using Magnetic-activated Cell Sorting (MACS) technique (Miltenyi Biotech, Bergisch Gladbach, Germany) and “EasySep Positive Selection Human PE Selection Kit (StemCell Technologies, (Vancouver, BC, Canada)” following the manufacturer’s protocol. Shortly, fresh tumor tissue samples were physically minced with a scalpel and exposed to enzymatic dissociation using 400 μg/ml Collagenase enzyme (GIBCO, New York, USA) at 37 °C for 3 h. Dissociated cells were filtered using a 70-μm cell strainer to get a single cell suspension. Cells were labeled with CD133/2-PE (Miltenyi Biotech clone AC133) antibody. After magnetic sorting, CD133 enriched (CD133+) and remaining (CD133−) cell populations from the same tissue samples were immediately washed and homogenized in “Lysis/Binding Buffer” of “mirVana miRNA Isolation Kit” (Ambion, Darmstadt, Germany) for further RNA isolation.
5
Multicomponent Phytochemical Cocktail Protocol
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BSG which consisted of seven different phytochemicals “curcumin, indol-3-carbinol, resveratrol, quercetin, C-Phycocyanin, genistein and gallic acid” was kindly provided by Dr. Mahdwa Raj (Proteigene, USA), Phosphate buffer saline, Collagenase enzyme (Gibco, CA, USA).
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