Trypsin without EDTA (Beyotime, Haimen, China) was used to collect the transfected cells when they reached 80%. After fixation with 75% pre-cooled ethanol at 4 °C overnight, the cell cycle phases (G0/G1, S, and G2/M) were analyzed using FACS flow cytometer after gently washing twice in 0.01% phosphate-buffered saline (PBS). The proliferative index (PI = G2M + S) was used to analyze results. Flow cytometry also used to identify SCAPs through cell surface makers. The transfected SCAPs were collected by trypsin and washed with PBS. The samples with different primary antibodies (CD29, CD34, CD45, CD73, CD90) were incubated for 15 min under dark conditions. Stained cells were analyzed after being rinsed twice with PBS.
Trypsin without edta
Manufactured by Beyotime
Sourced in China
Trypsin without EDTA is a proteolytic enzyme used for cell detachment and dissociation in cell culture applications. It functions by cleaving peptide bonds, primarily at the carboxyl side of lysine and arginine residues. This product does not contain EDTA, a common additive in some trypsin formulations.
Automatically generated - may contain errors
Lab products found in correlation
Annexin 5 fitc, by Beyotime (1 mentions)
Propidium iodide, by Beyotime (1 mentions)
Facscalibur system, by BD (1 mentions)
Annexin 5 fitc apoptosis kit, by Keygen Biotech (1 mentions)
Zombie violet fixable viability kit, by BioLegend (1 mentions)
Annexin 5, by BioLegend (1 mentions)
Flow cytometry, by BD (1 mentions)
Cell cycle detection kit, by Keygen Biotech (1 mentions)
5 protocols using trypsin without edta
1
Cell Cycle Analysis and SCAP Characterization
2
Apoptosis detection by flow cytometry
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After reoxygenation, cells were collected with trypsin without EDTA (Beyotime, Shanghai, China). After washing twice with ice-cold PBS, cells were resuspended in 200 μL of binding buffer, and 5 μL of AnnexinV-FITC and 5 μL of propidium iodide (Beyotime, Shanghai, China) were added, and the cells were incubated at room temperature for 15 min in the dark. An additional 400 μL of binding buffer was added and the cells were analyzed by flow cytometry.
3
Annexin V-FITC Apoptosis Assay
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An Annexin V-FITC Apoptosis Kit (KeyGen Biotech, Nanjing, China) was used to analyze cell apoptosis according to the manufacturer’s instructions. SMMC-7721 and HepG2 cells of logarithmic growth phase (1 × 105/ml) were seeded in six-well plates. Then, SMG9 NC or SMG9 siRNA was transfected into SMMC-7721 and HepG2 cells with Lipofectamine™ 3,000. Forty eight hours post-transfection, all cells (including cells in the supernatant) were harvested with trypsin without EDTA (Beyotime, Shanghai, China) and centrifuged at 1,200 rpm for 5 min. After washing with cold PBS twice, the cells were resuspended in 500 μl binding buffer and incubated with 5 μl Annexin V- FITC and 5 μl PI at room temperature for 15 min in the dark. Then, the samples were analyzed on a flow cytometer (BD FACS Calibur System; BD, United States).
4
Evaluating Cytotoxicity and Apoptosis
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For H22 cells, 2×10^4 cells were seeded in six-well plates with 1 mL complete RPMI 1640 medium containing 0.5% FBS and treated with Dox (0.5 μg/mL), TMPs (0.05 U), Dox-TMPs (0.05 U, containing 0.5 μg of Dox) or equivalent volume of PBS. After 24 hours of incubation in cell culture incubator, cells were harvested. For Huh7, HepG2 and Hepa1-6 cells, 2×10^4 cells were initially seeded in six-well culture plates with 1mL complete DMEM medium containing 10% FBS. After 6 hours of cell adhesion, the medium was replaced with 0.5% FBS-containing complete DMEM medium `emented with Dox (0.5 μg/mL), TMPs (0.05 U), Dox-TMPs (0.05 U, containing 0.5 μg of Dox) or equivalent volume of PBS. The cells were then cultured for an additional 44 hours before the supernatant and cells were collected after digestion with trypsin without EDTA (Beyotime, #C0205, Shanghai, China). Finally, all four cell types were stained with Annexin V (Biolegend, #640920, San Diego, CA, USA) and Zombie Violet™ Fixable Viability Kit (Biolegend, #423113, San Diego, CA, USA) and analyzed using the Sony ID7000 flow cytometer.
Cell apoptosis was also assessed using LDH Cytotoxicity Assay Kit. The drug treatment conditions were the same as mentioned above, and the detection procedure was performed following the manufacturer’s instructions.
Cell apoptosis was also assessed using LDH Cytotoxicity Assay Kit. The drug treatment conditions were the same as mentioned above, and the detection procedure was performed following the manufacturer’s instructions.
5
Cell Cycle Analysis by Flow Cytometry
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Cells after transfection were collected using trypsin without EDTA (Beyotime, China) and fixed with 75% alcohol overnight, then cells were incubated with RNase and PI for 30 min at RT and detected by Flow Cytometry (BD, USA) according to the manufacturer’s protocol of Cell Cycle Detection Kit (KeyGEN BioTECH).
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