SZ95 sebocytes were seeded into 6-well plates. Following exposure for 48 h with different concentrations of PM2.5 suspension, cells were collected using 0.25% trypsin and phosphate-buffered saline (PBS). Also, culture was centrifuged at 300 × g for 5 min at room temperature twice. After that precooled 90% ethanol was added. Then cells were resuspended at 4°C for 20 min and centrifuged at 300 × g for 5 min at room temperature. Cells were incubated with propidium iodide (PI) buffer [50 mg/ml containing ribonuclease A (50 ng/ml)] at room temperature and stained in the dark for 20 min at room temperature. Cell cycle distribution was analyzed using a BD Biosciences Flow Cytometer (BD Biosciences, San Jose, CA, USA).
Biosciences flow cytometer
Manufactured by BD
Sourced in United States
The BD Biosciences flow cytometer is a laboratory instrument designed for the analysis and sorting of cells and other microscopic particles. It utilizes the principles of flow cytometry to rapidly measure and analyze multiple physical and chemical characteristics of individual cells or particles as they flow in a fluid stream through a beam of light.
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5 protocols using biosciences flow cytometer
1
Cell Cycle Analysis of PM2.5-Exposed Sebocytes
2
Sanguinarine Modulates Platelet Ca2+ Flux
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Intracellular Ca2+ flux was measured by flow cytometry [19 (link)]. Washed human platelets (5 × 107/mL) were labeled with 1 μM Fluo-3AM at 37 °C for 30 min, then incubated with sanguinarine (1, 2.5 and 5 μM) or DMSO (control) for 10 min. After collecting a baseline reading for 20 s, collagen (2 µg/mL) and 2 mM extracellular calcium were added to the FACS tube, and the change in fluorescence intensity was recorded on a BD Biosciences flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and analyzed using Flowjo V10 (BD Biosciences, Franklin Lakes, NJ, USA). The fitting curve was plotted using SigmaPlot software 14.0 (Systat Software, Inc., San Jose, CA, USA).
3
Apoptosis Assay of NSC-34 Cells
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Transfected or un-transfected NSC-34 cells were plated at 5 × 105 cells per well in 24-well plates and then conducted LPS or/and TSIIA treatment for 24 h. After that, cells were washed using phosphate buffered saline (PBS, Solarbio, Beijing, China), stained with Annexin V labeled by fluorescein isothiocyanate (FITC) (BD Biosciences, Heidelberg, Germany) and propidium iodide (PI, Life Technologies) for 15 min, and analyzed within 1 h. These experiments were done in triplicate and 10,000 gated events per sample were counted using the BD Biosciences flow cytometer and AccuriC6 software (BD Biosciences).
4
Adipose-Derived Stem Cell Isolation and Characterization
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Patients (5 female donors, 45.4 ± 5.413 years old) from Plastic Surgery Hospital, Chinese Academy of Medical Sciences, and Peking Union Medical College (CAMS & PUMC) were recruited to provide healthy adipose tissues following selective abdominal liposuction. All donors signed informed consent forms. After being washed twice with PBS, fat tissues were digested in a shaker at 37 °C for 45 min with the same volume of 0.2% (w/v) type I collagenase (Sigma-Aldrich, Darmstadt, Germany). Upon centrifugation at 400× g for 5 min, the supernatant was discarded, and the precipitation was resuspended in α-MEM. The cell suspension was filtered through a 70 μm nylon filter and then cultured in complete α-MEM (containing 10% FBS and 1% PSN) on a 10 cm culture plate. ADSCs were cultured at 37 °C in a humidified atmosphere of 5% CO2, and the media were changed every three days. At 80% confluence, the cells were defined as passage 1 and re-plated. Passages 2–3 were used for the following experiments. ADSCs were characterized by multilineage differentiation (Cyagen Bioscience, Guangzhou, China) and analysis of the expression of CD73, CD90, CD105, CD34, CD11b, CD19, and CD45 (BD Biosciences, Franklin Lakes, NJ, USA) using a BD Biosciences flow cytometer and Flow Jo software (version 10.8.1, BD Biosciences, Franklin Lakes, NJ, USA).
5
SPAG9 Silencing Impacts Apoptosis in HepG2 Cells
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After transfection with SPAG9-targeted siRNA or the control siRNA, HepG2 cells were harvested, washed twice with PBS, and fixed in 70% ethanol at 4˚C overnight. Cells were incubated with propidium iodide (PI) at room temperature for 1 h and were analyzed by flow cytometry using a BD Biosciences flow cytometer (BD Biosciences, San Jose, CA, USA).
Cell apoptosis assay. Apoptotic cells were distinguished from normal cells using an Annexin V-FITC/PI apoptosis kit for flow cytometry (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. After transfection with SPAG9 siRNA or siRNA control, HepG2 cells were harvested and washed twice with cold PBS and then incubated with 5 µl FITC-Annexin V and 1 µl PI working solution (100 µg/ml) for 15 min in the dark at room temperature. Cellular fluorescence was measured by flow cytometry.
Statistical analyses. Statistical analyses were performed using SPSS 17.0 (SPSS, Inc., Chicago, IL, USA). Data are presented as the means ± standard deviations (SD). Statistical analyses were performed with one-way ANOVA, two-way ANOVA and repeated measures ANOVA as appropriate. Bonferroni was used as a post hoc test in one-way ANOVA. The statistical significance level was set at P<0.05 (two-sided).
Cell apoptosis assay. Apoptotic cells were distinguished from normal cells using an Annexin V-FITC/PI apoptosis kit for flow cytometry (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. After transfection with SPAG9 siRNA or siRNA control, HepG2 cells were harvested and washed twice with cold PBS and then incubated with 5 µl FITC-Annexin V and 1 µl PI working solution (100 µg/ml) for 15 min in the dark at room temperature. Cellular fluorescence was measured by flow cytometry.
Statistical analyses. Statistical analyses were performed using SPSS 17.0 (SPSS, Inc., Chicago, IL, USA). Data are presented as the means ± standard deviations (SD). Statistical analyses were performed with one-way ANOVA, two-way ANOVA and repeated measures ANOVA as appropriate. Bonferroni was used as a post hoc test in one-way ANOVA. The statistical significance level was set at P<0.05 (two-sided).
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