hADSCs were harvested and fixed with 70% ethanol. After extensive washing with HBSS, the cells were suspended in HBSS containing RNase A and PI (Sigma). The cells were incubated for 30 min at 37 °C before flow-cytometric analysis using a FACS Canto II instrument (BD, San Jose, CA, USA) and the acquired data were analyzed using the FlowJo software (Tree Star, Ashland, OR, USA).
Rnase a and pi
Manufactured by Merck Group
Sourced in United States
RNase A and PI are laboratory reagents used for the isolation and purification of RNA. RNase A is an enzyme that catalyzes the hydrolysis of single-stranded RNA, while PI (Propidium Iodide) is a fluorescent dye that binds to nucleic acids. These products are commonly used in various molecular biology and life science applications, such as RNA extraction, gene expression analysis, and cell biology studies.
Automatically generated - may contain errors
Lab products found in correlation
Annexin 5, by BD (2 mentions)
Propidium iodide, by BD (2 mentions)
Facscanto 2, by BD (1 mentions)
Annexin 5 fitc apoptosis detection kit, by Roche (1 mentions)
Nonidet p 40, by AppliChem (1 mentions)
3 4 5 dimethylthiazol 2 yl 5 3 carboxymethoxyphenyl 2 4 sulfophenyl 2h tetrazolium mts reagent, by Promega (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Quantitect sybr green pcr kit, by Qiagen (1 mentions)
Facscalibur, by BD (1 mentions)
Facs c6, by BD (1 mentions)
Binding buffer, by BD (1 mentions)
6 protocols using rnase a and pi
1
Flow Cytometry Analysis of hADSCs
2
Comparative Evaluation of NaB, Cur, and Pip
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NaB was purchased from National Boron Research Institute-BOREN (Ankara, Turkey). NaB stocks were prepared as 10 mg/mL in completed media using vortex and all solutions were filtered with a 0.22 μm filter before use. Cur and Pip were purchased from Sigma-Aldrich (St. Louis, MO, USA) and resuspended in dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St Louis, MO) to prepare a stock solution of 50 mM and 150 mM, respectively. The main stock solution was filtered through a 0.22 μm filter and kept at − 20 °C for long time storage.
MTS reagent ([3-(4,5-dimethylthiazol-2-yl)-5- (3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) was purchased from Promega (Southampton, UK). Annexin V-FITC Apoptosis Detection Kit was purchased from Roche (Germany). RNase A and PI were obtained from Sigma-Aldrich (Germany). Nonidet P-40 was obtained from AppliChem (Germany). RNeasy Mini kit, Sensiscript Reverse Transcription PCR Kit and QuantiTect SYBR Green PCR kit were supplied by Qiagen (United States).
MTS reagent ([3-(4,5-dimethylthiazol-2-yl)-5- (3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) was purchased from Promega (Southampton, UK). Annexin V-FITC Apoptosis Detection Kit was purchased from Roche (Germany). RNase A and PI were obtained from Sigma-Aldrich (Germany). Nonidet P-40 was obtained from AppliChem (Germany). RNeasy Mini kit, Sensiscript Reverse Transcription PCR Kit and QuantiTect SYBR Green PCR kit were supplied by Qiagen (United States).
3
Cell Cycle Analysis by Flow Cytometry
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Briefly, 24 h after transfection, cells were harvested and fixed in 70% ethanol and stored at −20°C for 6 h. Cells were then washed twice with ice-cold PBS and incubated with RNase A and PI (Sigma) for 30 min in the dark at room temperature. Cell cycle assays were performed using a flow cytometer (BD FACSCalibur, BD Bioscience). Cell cycle phase analysis was performed using the Modifit cell cycle analysis software (Becton Dickinson).
4
Flow Cytometric Analysis of Apoptosis and Cell Cycle
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The Alexa Fluorescence488 (AF488)-conjugated monoclonal antibodies specific for α-SMA (R&D Systems, Minneapolis, MN, USA), a rabbit anti-FAP polyclonal antibody (Abcam, Cambridge, MA, USA) and a FITC-conjugated anti-rabbit polyclonal antibody were used (eBioscience, San Diego, CA, USA). The appropriate isotype-matched antibodies were used as controls (R&D Systems). For the apoptosis assay, THP-1 and K562 cells (5 × 105) were stained with Annexin-V and propidium iodide (PI) for apoptosis detection (BD, Franklin Lakes, New Jersey, USA) according to the recommended protocol on a Becton Dickinson Flow Cytometer. For cell cycle analyses, THP-1 cells were treated with RNase A and PI (Sigma, St Louis, MO, USA). The measurements were made using a flow cytometer (Beckman, Urbana, IL, USA).
5
Apoptosis and Cell Cycle Analysis
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After treatment with DMSO or T-96 at 37 °C for 3 days, cells were harvested and washed with cold PBS buffer and then resuspended in 100 μL of binding buffer (BD, USA). Then, the cells were incubated with FITC-labelled Annexin V (BD, San Jose, CA, USA) and propidium iodide (PI, BD, USA) at room temperature for 15 min and analysed via flow cytometry as previously described62 (link). For the cell cycle assay, cells were harvested and fixed in ice-cold 70% ethanol at 4 °C for 1 day. After being washed and resuspended in PBS buffer, the cells were incubated with PI and RNase A (Sigma Aldrich, USA) at 37 °C for 1 h. All samples were analysed using a FACS C6 (BD, USA) with Cell Quest software.
6
Cell Cycle Analysis by Flow Cytometry
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Cells were cultured for 24 hours and then digested with 0.25% trypsin (without EDTA) into individual cells. The cells were collected by centrifugation, and the supernatant was discarded. The cells were washed twice with precooled PBS. Three milliliters of precooled 70% ethanol was added to the cell precipitate, and the cells were fixed overnight at 4°C. The cells were collected by centrifugation, washed twice with 3 mL of PBS, and then stained with PI and RNase A (Sigma). The cells were then incubated at 4°C for 30 min without light. PI fluorescence intensity was analyzed by a flow cytometer using the FL-2 channel.
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