The pro-apoptotic effect of TF was evaluated by DAPI staining and flow cytometry assay using annexin-V/PI staining. For DAPI staining, B16F10 cells were seeded into 24-well plates at 1.5 ×105 cells/well and treated with TF at low, medium, high concentrations for 24h, followed by fixation with 4% paraformaldehyde in PBS for 30 min at room temperature and staining with DAPI for 10 min in dark. After wash for three times, the cells were observed using five coverslips under Carl Zeiss fluorescence microscope (Göttingen, Germany) and the apoptotic cells were counted. For flow cytometry assay, B16F10 cells were seeded into 6-well plates at 3×105 cells/well for 24 h and treated with TF at low, medium, high concentrations for 72 h. Afterwards, the cells were washed twice and labeled with annexin V-fluorescein isothiocyanate solution and PI in binding buffer. Fluorescence intensity of the cells was detected by BD C6 flow cytometry (CA, USA). The early apoptotic and late apoptotic cell rates (%) were calculated.
C6 flow cytometry
Manufactured by BD
Sourced in United States
The C6 flow cytometry is a laboratory instrument used for the analysis and sorting of cells and other particles. It is designed to measure the physical and chemical characteristics of these cells or particles as they flow in a fluid stream through a beam of light. The C6 flow cytometer provides quantitative data on parameters such as cell size, granularity, and fluorescence, allowing researchers to identify and analyze different cell populations.
Automatically generated - may contain errors
Lab products found in correlation
Fluorescence microscope, by Zeiss (2 mentions)
Ti e a1, by Nikon (1 mentions)
Annexin 5 fitc pi apoptosis detection kit, by Keygen Biotech (1 mentions)
Jc 1 assay, by Beyotime (1 mentions)
Ferroorange, by Dojindo Laboratories (1 mentions)
Annexin 5 apc 7 add apoptosis detection kit, by Keygen Biotech (1 mentions)
Ebioscience annexin 5 fitc apoptosis detection kit, by Thermo Fisher Scientific (1 mentions)
Live dead fixable near ir dead cell stain kit, by Thermo Fisher Scientific (1 mentions)
Fitc annexinv pi apoptosis detection kit, by Beyotime (1 mentions)
Microtiter plate reader, by Thermo Fisher Scientific (1 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Fluorescein isothiocyanate (fitc), by Jackson ImmunoResearch (1 mentions)
Lms 710 confocal microscopy, by Zeiss (1 mentions)
Anti his antibody, by Merck Group (1 mentions)
23 protocols using c6 flow cytometry
1
Evaluating Apoptotic Effects of TF in B16F10 Cells
2
Nanoparticle Cellular Uptake Kinetics
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Flow cytometry analysis: HaCaT cells were cultured at 1 × 105 cells well−1 in 12 well plates. When the cells properly adhered, the medium was removed and FITC-loaded PLGA nanoparticle suspension (100 µg ml−1) was incubated for 1, 2, 4 and 8 h. After different exposure times, cells were washed twice with phosphate buffered saline (PBS) and harvested using trypsin–EDTA (0.25% v/v). The results were recorded using a BD C6 flow cytometry (FACS) with a 488 nm laser. For each experiment, a total of 10 000 cells were gated per sample and each sample was performed in triplicate.
Confocal laser scanning microscopy: laser confocal microscopy (Ti-E A1, Nikon, Japan) was used to qualitatively analyse the localization of fluorescent nanoparticles in HaCaT cells. The cells were seeded at 5 × 104 cells well−1 in 24-well plates containing glass coverslips. After incubation with fluorescent nanoparticles for a certain period of time, the cells were washed gently with PBS three times and fixed with 4% paraformaldehyde for 20 min. Finally, cells were stained with 4,6-diamidino-2 phenylindole (DAPI) for 5 min.
Confocal laser scanning microscopy: laser confocal microscopy (Ti-E A1, Nikon, Japan) was used to qualitatively analyse the localization of fluorescent nanoparticles in HaCaT cells. The cells were seeded at 5 × 104 cells well−1 in 24-well plates containing glass coverslips. After incubation with fluorescent nanoparticles for a certain period of time, the cells were washed gently with PBS three times and fixed with 4% paraformaldehyde for 20 min. Finally, cells were stained with 4,6-diamidino-2 phenylindole (DAPI) for 5 min.
3
Apoptosis Quantification by DAPI and Annexin V/PI
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Cell apoptosis was determined by DAPI staining and annexin-V/PI staining-based flow cytometry. For DAPI staining, A375 cells were seeded into 96-well plates and treated with TF at low, medium, and high concentrations for 24 h, followed by fixation with 4% paraformaldehyde in PBS for 30 min at room temperature and staining with DAPI for 10 min in dark. After thrice wash, cells were observed using five coverslips under Carl Zeiss fluorescence microscope (Göttingen, Germany) and the apoptotic cells were counted. Flow cytometry was conducted according to the manufacturer′s instruction. Briefly, A375 cells were seeded into 6-well plates at 3×105 cells/well for 24 h and treated with TF at low, medium, and high concentrations for another 48 h. Afterwards, the cells were washed twice and labeled with annexin V-fluorescein isothiocyanate solution and PI in binding buffer. Fluorescence intensity of the cells was detected by BD C6 flow cytometry (CA, USA). The analysis was replicated and the early apoptotic and late apoptotic cell rates (%) were calculated.
4
Flow Cytometry of Alveolar Cells
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Flow cytometry of alveolar cells was preformed as previously described[30 (link)]. BALF cells were resuspended in wash buffer (1% BSA-PBS), fluorescently labeled antibodies were incubated at 4°C in dark for 20 minutes, and washed twice with ice wash buffer. Cells were detected by BD C6 Flow Cytometry and the data were analyzed by C6 software.
5
Cell Cycle Analysis by Flow Cytometry
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Cell flow cytometry was performed 48 h after siRNA transfection. Cells were collected using EDTA-free trypsin, washed twice using 0.5 ml PBS, suspended in cold 70% ethanol, and stored in 4 °C overnight. Ethanol was removed and cells were re-suspended in PBS the next day followed by 0.05 mg/ml Propidium Iodide (PI) addition. Cells were kept in darkness on ice for 30 min before being sent to BD C6 flow cytometry. Analysis was performed using the flowjo software.
6
Apoptosis Assay for Human Granulosa Cells
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The human primary granulosa cells were seeded in 12-well plates at the density of 3–4 × 105cells/well, conventionally cultured for 24 h, and then starved in the starvation culture medium for 24 h. The adherent granulosa cells were collected by trypsin digestion after treatment with 10 μM Baicalin for 72 h, according to the instructions of an Annexin V-FITC/PI Apoptosis Detection Kit (KeyGen Biotech, Nanjing, People’s Republic of China). Briefly, the cells were collected using a trypsin digestion solution without EDTA, washed twice with pre-cooled PBS, and centrifuged at 2000 rpm for 5 min. Subsequently, the cell pellet was suspended in 500 μL binding buffer, and 5 μL Annexin-FITC was added and mixed well. Then, 5 μL of propidium iodide was added and mixed well, and the mixture was kept in the dark at 22–25 °C for 5–15 min, followed by cell apoptosis detection using BD C6 Flow Cytometry (BD Biosciences, San Jose, CA, USA) within 1 h.
7
Flow Cytometry Analysis of CD44
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The anti-CD44 antibody (eBioscience, USA) was used for flow cytometry analysis according to the guidance of manufacturer. Samples were analyzed using C6 flow cytometry (Becton Dickinson, USA) and data was analyzed by FlowJo software (TreeStar, USA).
8
Mitochondrial Membrane Potential Assay
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Mitochondrial membrane potential (MMP) was assessed using the JC-1 assay (Beyotime, C2006, China). Following the instructions, the JC-1 working solution was prepared. After the cell collection from each group, they were thoroughly mixed with 500 μL of the JC-1 working solution, and incubated at 37°C for 20 minutes. Then, the cells were washed twice with precooled 1× JC-1 assay Buffer and then tested by a C6 flow cytometry (Becton Dickinson, USA).
9
Quantify Cellular Iron Levels
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To measure the Fe2+ levels in different groups, FerroOrange (F-374, Dojindo, China) was employed. Cells were collected into 1.5 mL ep tubes, washed with non-serum RPMI-1640, and then incubated at 37°C in an incubator with 1 µM FerroOrange for 30 minutes before being tested by a C6 flow cytometry (Becton Dickinson, USA).
10
Annexin V-APC/7-ADD Apoptosis Assay
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Annexin V-APC/7-ADD Apoptosis Detection Kit (KeyGen, Nanjing, China) was used to determine cell apoptosis. The cells were suspended with 100 μl of binding buffer (10 mM HEPES/NaOH, 140 mM NaCl, 2.5 mM CaCl2, pH 7.4) and stained with 5 μl of APC-conjugated Annexin V and 5 μl of 7-ADD for 15 min at room temperature in dark and then 400 μl binding buffer was added. Apoptotic cells were analyzed by C6 flow cytometry (BD Biosciences, San Diego, CA, USA).
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