For the detection of intracellular calcium concentration, BMMSCs were incubated with 5 μM Fluo‐3/AM dye (Invitrogen, Life Technology) for 60 min at 37°C, followed by washing with PBS for three times. After digestion by trypsin, 2 × 105 cells were counted with PBS washing for two times. Then, cells were subjected to flow cytometric analysis (Beckman Coulter). Data are presented as fluorescence intensity.
Flow cytometric analysis
Manufactured by Beckman Coulter
Sourced in United States
Flow cytometric analysis is a technique that utilizes a flow cytometer to analyze the physical and chemical characteristics of cells or particles in a fluid sample. The flow cytometer measures various parameters, such as cell size, granularity, and fluorescence intensity, as the cells or particles pass through a laser beam in single file.
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Lab products found in correlation
Fluo 3 am dye, by Thermo Fisher Scientific (1 mentions)
Anti cd3 antibody, by Thermo Fisher Scientific (1 mentions)
Celltrace cfse dye, by Thermo Fisher Scientific (1 mentions)
Interleukin 2 (il 2), by R&D Systems (1 mentions)
Annexin 5 fluorescein isothiocyanate fitc kit, by BD (1 mentions)
Novocyte, by Agilent Technologies (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
Jc 1 probe, by Beyotime (1 mentions)
Nocodazole, by Merck Group (1 mentions)
Annexin 5 fitc, by BD (1 mentions)
10 protocols using flow cytometric analysis
1
Intracellular Calcium Quantification
2
Lymphocyte-HeLa Co-culture for T Cell Proliferation
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A lymphocyte-HeLa cells co-culture system was performed to determine whether BN@HM-OVA can reverse the inhibition of T cell proliferation caused by IDO [19 (link)]. The lymphocytes generated from C57BL/6 mice spleen were seeded in a 96-well plate at a density of 5 × 104 cells per well and cultured in medium containing 100 ng/mL anti-CD3 antibody (eBioscience, Thermo Fisher Scientific) and 10 ng/mL human recombinant interleukin-2 (IL-2, R&D Systems, Shanghai, China) for 3 days. After incubation for 3 days, lymphocytes were stained with CellTraceTM CFSE dye (ThermoFisher Scientific). The HeLa cells (1 × 104 cells per well) were stimulated with IFN-γ for 48 h and then co-cultured with lymphocytes. Various formulations of PBS, HM, free NLG-919 (20 μM), free BLZ-945 (1 μM), free OVA (25 μg/mL) and BN@HM-OVA having the same concentration of drugs and antigen were added to the co-culture system. After 3 days co-culture, T cell proliferation was measured by flow cytometric analysis (Beckman).
3
Quantification of Autophagy Vacuoles via Acridine Orange Staining
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Formation of AVO is a characteristic feature of autophagic cells [3 (link)]. We performed AO staining to detect and quantify AVO after treatment with different doses of SS. Briefly, overnight grown cells were exposed to 0.1, 0.5, 1.0, and 2.0 mM SS for 24 h in slide cultures and then incubated with AO (1 μg/ml) for 15 min for staining. Another set of SS unexposed cells were used as control (CTL). After washing thrice with 1x phosphate-buffered saline (PBS, pH 7.4), all slides containing the AO stained cells were then observed under the fluorescence microscope to detect the formation of AVO. We also performed flow cytometric analysis (Beckman Coulter, Fullerton, CA, USA) after AO staining, as described above, of both GSC and SNB19 cells. In flow cytometric analysis of the AO stained cells, cytoplasm and nucleolus in non-autophagic cells showed green fluorescence (500–550 nm, FL-1 channel) whereas AVO in autophagic cells (quadrant A1) showed bright red fluorescence (650 nm, FL-3 channel). Red fluorescence intensity is directly proportional to the number of AVO in autophagic cells. Therefore, on the basis of red fluorescence intensity, formation of AVO in autophagic cells can be determined.
4
Apoptosis and Lymphocyte Subset Analysis
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An annexin-V-fluorescein isothiocyanate (FITC) kit (BD Biosciences, San Jose, CA, United States) was used to measure the proportion of cells undergoing apoptosis. BRL 3A cells were treated with AT-1, collected, resuspended in 500 μl augmented binding buffer, and incubated for 15 min at 25°C in the dark in a reagent mix comprising 5 μl of the annexin-V-FITC conjugate and 10 μl of PI. The samples were analyzed using a flow cytometer (Novocyte, Agilent, United States) to determine the cell percentage at various phases.
For detecting lymphocyte subsets, 100 μl of the blood sample was stained with 10 μl of FITC-conjugated CD3, PE-conjugated CD4, and APC-conjugated CD8 reagent (Beckman Coulter), followed by incubation for 15 min in the dark for lysis of red blood cells. After washing, the cells were resuspended and subjected to flow cytometric analysis (Beckman). Lymphocytes were defined with their forward and side scatter characteristics. T lymphocytes were identified (CD3+) and then subdivided into CD4+ or CD8+ populations.
For detecting lymphocyte subsets, 100 μl of the blood sample was stained with 10 μl of FITC-conjugated CD3, PE-conjugated CD4, and APC-conjugated CD8 reagent (Beckman Coulter), followed by incubation for 15 min in the dark for lysis of red blood cells. After washing, the cells were resuspended and subjected to flow cytometric analysis (Beckman). Lymphocytes were defined with their forward and side scatter characteristics. T lymphocytes were identified (CD3+) and then subdivided into CD4+ or CD8+ populations.
5
Apoptosis Analysis by Annexin-V and TUNEL
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Cell apoptosis analysis was determined by Annexin-VV and PI Double-Staining assays (Annexin-V-FITC Apoptosis Detection kit, Bipec Biopharma, USA) and Terminal Uridine Nick End-Labeling assays (TUNEL, Roche, German). For Annexin-V and PI Double-Staining assays, cells were rinsed with PBS and harvested after 24-h starvation. Flow cytometric analysis (Beckman Coulter, USA) was performed following the double staining of FITC-conjugated Annexin-V and PI according to manufacturer's instruction. For TUNEL assays, cells were grown on coverslips overnight, followed with transfection for 12 h. After starvation for 48 h, TUNEL and DAPI staining were performed following manufacturer's instructions. Images were obtained using fluorescence microscopy, and the apoptosis index of cells was analyzed using Image-Pro Plus 6.0 software (Media Cybernetics, Inc.).
6
Mitochondrial Membrane Potential Assay
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The ΔΨm of hPDLSCs was measured using a mitochondrial membrane potential assay kit with JC-1 probe (Beyotime). When live cells are incubated with JC-1, the mitochondria are driven by the ΔΨm and JC-1 is rapidly taken up, raising the JC-1 concentration and the formation of aggregates (J-aggregates) within the mitochondria, which provoke red fluorescence. However, JC-1 does not accumulate in depolarized mitochondria with low ΔΨm and remains in the cytoplasm as monomers, which emit green fluorescence. hPDLSCs were incubated in JC-1 working solution at 37°C for 20 min, then were washed twice with wash buffer and measured by flow cytometric analysis (Beckman Coulter) or using a fluorescent microscope (Carl Zeiss).
7
Cell Cycle Analysis of TACC3-expressing Bladder Cancer Cells
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Bladder cancer cell lines that stably express TACC3 were treated with nocodazole (100 ng/ml, Sigma-Aldrich, St Louis, MO, USA) for 16 h. Cells were harvested by trypsinization and fixed in ice-cold 70% ethanol overnight. Cell-cycle distributions were measured by staining with propidium iodide, which was followed by flow cytometric analysis (Beckman).
8
Evaluating MSC Viability under Hypoxia/Reoxygenation
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MSCs cocultured with H/R model followed treated with GW4869 for 12 h. Cells were collected and resuspended in PBS or medium, centrifugated at 1000 rpm for 5 min. Next, 200 μL binding buffer resuspended the sediment and blended. Cells were incubated with 5μL Annexin V-FITC/PI for 10 min in dark, and centrifugated at 1000 rpm for 5 min. The sediment was resuspended with 200 μL binding buffer again followed by added 5 μL PI before detection. Samples were detected by Flow Cytometric Analysis (Beckman Coulter, CA, USA).
9
Quantifying Cell Apoptosis by Flow Cytometry
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C6 cells were seeded at 4.0×105 cells/well in 6-well plates and treated with FLT for 24 h, and then washed twice with cold PBS and re-suspended at a concentration of 1×106 cells/ml. Next, 5 μl FITC-Annexin V and 10 μl PI (both from BD Biosciences) were added into 100 μl of the solution. After that, the cells were mixed gently and incubated for 15 min at room temperature in the dark. Finally, 1 ml PBS was added and the samples were detected by flow cytometric analysis (Beckman Coulter, Brea, CA, USA) within 1 h.
10
Evaluating Apoptosis in HSC-BMSC Co-culture
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HSCs were cultured in a specific medium with 10 % FBS and 1 % Pen-Strep solution. A total of 2 × 105 HSCs were seeded in 6-well plates. The cells were grown overnight in a serum-deprived medium. Afterward, the activated phenotype of HSCs was formed by incubation with TGF-β1 (10 ng/mL) for 24 h. Co-culturing of activated HSCs with Mi-BMSCs or BMSCs was performed for 24 h. For the apoptosis assays, cells were collected and stained with 5 μL Annexin V-FITC and 10 μL propidium iodide (PI) solution. Cells were incubated for 5 min at room temperature and analyzed by flow cytometric analysis (Beckman, USA).
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