The cardiac-differentiated hESCs were isolated and analyzed by flow cytometry. Approximately 1 × 105 cells per sample were fixed with 4% paraformaldehyde (Biosharp, China) and washed with staining buffer (1% fetal bovine serum in PBS) (Beyotime, China). The cells were then permeabilized with 100% cold methanol and incubated with primary antibodies, followed by the corresponding secondary antibody. The primary antibodies contained rabbit anti-TET2 (diluted 1:500, Abcam, ab94580) and rabbit anti-cTnT (diluted 1:500, Abcam, ab209813). The secondary antibodies used were Anti-rabbit IgG (H + L), F(ab’)2 Fragment (Alexa Fluor® 488 Conjugate) (diluted 1:1000, Cell Signaling Technology, #4412) and Anti-Rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody (Alexa Fluor™ 647) (diluted 1:1000, Invitrogen, A-21244). As shown in Additional file 1 : Fig. S6, cell gating was performed by using isotype controls, such as Rabbit (DA1E) mAb IgG XP® Isotype Control (Alexa Fluor® 647 Conjugate) (diluted 1:1000, Cell Signaling Technology, #2985) and Rabbit (DA1E) mAb IgG XP® Isotype Control (Alexa Fluor® 488 Conjugate) (diluted 1:1000, Cell Signaling Technology, #2975). The samples were acquired using Amnis Image StreamX MarkII flow cytometer (Merck & Millipore, Germany) and analyzed with IDEAs.6.2 software.
Staining buffer
Manufactured by Beyotime
Sourced in China
Staining buffer is a laboratory solution used to prepare samples for staining and visualization procedures. It serves to maintain the sample's pH and ionic balance, creating an optimal environment for effective staining and imaging.
Automatically generated - may contain errors
Lab products found in correlation
Rnase a, by Beyotime (2 mentions)
Propidium iodide, by Beyotime (2 mentions)
Amnis imagestreamx mark 2 flow cytometer, by Merck Group (1 mentions)
Anti rabbit igg h l cross adsorbed secondary antibody alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Biosharp (1 mentions)
Fetal bovine serum (fbs), by Beyotime (1 mentions)
Ab94580, by Abcam (1 mentions)
Ab209813, by Abcam (1 mentions)
Binding buffer, by Keygen Biotech (1 mentions)
Annexin 5 fitc, by Keygen Biotech (1 mentions)
Multicycle software, by Beckman Coulter (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Fluorescence microscope, by Beyotime (1 mentions)
Accuri c6, by BD (1 mentions)
Novocyte flow cytometer, by Agilent Technologies (1 mentions)
Annexin 5 fluorescein isothiocyanate fitc, by Beyotime (1 mentions)
6 protocols using staining buffer
1
Flow Cytometric Analysis of Cardiac-Differentiated hESCs
2
Cell Cycle Analysis and Apoptosis Detection in H9c2 Cells
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For cell cycle analysis, H9c2 cells were washed with PBS and fixed in 70% ethanol. The cells were incubated with 25 µl propidium iodide (PI)/10 µl RNaseA in staining buffer (Beyotime Institute of Biotechnology) at 37°C for 30 min in the dark. After incubation, the cells were subjected to cytometric analysis. For apoptosis detection, the cells were treated with 5 µl Annexin V-FITC/5 µl propidium iodide (PI) in binding buffer (KeyGen, Nanjing, China) for 15 min before analysis.
3
Cell Cycle Analysis by Flow Cytometry
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Cells were collected, fixed with 70% alcohol at 4 °C for half an hour, and then washed twice with cold phosphate-buffered saline (PBS). Then, a mixture of RNaseA (Beyotime), propidium iodide (PI, Beyotime) and staining buffer (Beyotime) was added to the cells, which were incubated at 37 °C for half an hour in the dark following the manufacturer’s instructions. A FACSCalibur flow cytometer (BD, USA) was used to determine the cell cycle distribution. The results were obtained using MultiCycle software (Beckman Coulter, USA).
4
Mitochondrial Membrane Potential Assay
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For mitochondrial membrane potential assays, the cells were seeded in a 6-well plate at a density of 15×104 cells/well. A total of 24 h after the start of the inoculation, the cells were stained for 30 min with 15 µmol/l5,5′, 6,6′-tetrachloro-1,1′, 3,3′-tetraethyl-imidacarbocyanineiodide (JC-1; Beyotime Institute of Biotechnology) in cell culture media at 37°C followed by two washes with staining buffer (Beyotime Institute of Biotechnology) prior to analysis using a fluorescence microscope (magnification, ×400). The ratio between green and red fluorescence signals served as a parameter for the mitochondrial membrane potential (ΔΨm) and is independent of the mitochondrial mass.
5
Annexin V-FITC Apoptosis Assay of Leukemia Cells
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Leukemia cells were cultured in a six-well plate (2×105 cells/well) and then infected with oVV or oVV-ING4 at 10 MOI. After incubation for 48 hours at 37°C, the cells were rinsed two times with PBS, suspended in 300 µL of binding buffer, and treated with 5 µL of Annexin V-fluorescein isothiocyanate (FITC) and 10 µL of propidium iodide (PI) (Beyotime, Jiangsu, China) for 15 minutes in the dark at room temperature. Subsequently, the cells were studied using NovoCyte Flow Cytometer (ACEA Biosciences, San Diego, CA, USA) using an inbuilt software (Novo Express) and by flow cytometry (Accuri C6; BD Biosciences, San Jose, CA, USA). Fluorescence activated cell sorter (FACS) analysis was applied to detect the cell-cycle status of leukemia cells infected with oVV or oVV-ING4. Cells were harvested, rinsed twice in cold PBS, re-suspended in cold 75% alcohol, and fixed overnight at −20°C. Then they were centrifuged and rinsed twice in cold PBS. Later, they were incubated in 500 µL of staining buffer (Beyotime) added with 25 µL of 20× PI and 10 µL of 50× RNase A in the dark for 30 minutes at 4°C, followed by examination immediately using fluorescence microscope.
6
Cell Cycle and Apoptosis Analysis
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A cell-cycle and apoptosis analysis kit with propidium iodide staining reagent (Beyotime) was used for flow cytometric analysis. Cells were harvested by trypsinization, washed once with cold PBS, and suspended in 70% ethanol; cells were fixed by paraformaldehyde overnight with rotation. Before staining, the cells were washed with PBS. Then, cells were incubated with staining buffer (Beyotime) containing propidium iodide and RNase A in a 37 °C water bath for 0.5 h and then analyzed with flow cytometry.
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