Brain endothelial cell (BEC) expression of claudin-5 and ZO-1 was examined as described previously [36 (link)]. Briefly, BECs derived from the β4-EC-KO or littermate control mice were first passaged onto laminin-coated six-well plates. Upon reaching confluence, the cells were treated with either 10 ng/ml TNF-α or IFN-γ (both from R&D) for 48 h and then removed and cellular expression of claudin-5 and ZO-1 analyzed by flow cytometry using rabbit polyclonal anti-claudin-5 or anti-ZO-1 antibodies followed by anti-rabbit Alex Fluor 488 secondary (both from Invitrogen). As the claudin-5 and ZO-1 antibodies are directed against intracellular epitopes, the cells were first fixed and then permeabilized using the Cytofix/Cytoperm kit (BD Pharmingen) and all subsequent incubations were performed in the cytoperm buffer. The fluorescent intensity of labeled cells was analyzed with a Becton Dickinson FACScan machine, with 10,000 events captured for each condition. Each experiment was repeated a minimum of four times and the data expressed as mean ± SEM fluorescent intensity. Statistical significance was assessed by using the Student’s paired t test, in which p < 0.05 was defined as statistically significant.
Facscan machine
Manufactured by BD
Sourced in United States
The FACScan is a flow cytometry instrument manufactured by BD. It is designed to analyze and sort cells based on their physical and fluorescent characteristics. The FACScan utilizes a laser to excite fluorescent-labeled cells, and it then detects and measures the emitted light signals to provide quantitative data about the cells' properties.
Automatically generated - may contain errors
Lab products found in correlation
Cellquest software, by BD (2 mentions)
Pi rnase staining buffer, by BD (2 mentions)
Alex fluor 488 secondary, by Thermo Fisher Scientific (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
Tnf α, by R&D Systems (1 mentions)
Ifn γ, by R&D Systems (1 mentions)
Evos fl cell imaging system, by Thermo Fisher Scientific (1 mentions)
Facsvantage se, by BD (1 mentions)
Orca 100 ccd camera, by Hamamatsu Photonics (1 mentions)
Dmr wide field epifluorescence microscope, by Hamamatsu Photonics (1 mentions)
Openlab version 3, by PerkinElmer (1 mentions)
Sytox green, by Thermo Fisher Scientific (1 mentions)
Annexin 5 pi double staining kit, by Dojindo Laboratories (1 mentions)
Mitosox red, by Thermo Fisher Scientific (1 mentions)
Pe conjugated anti human ifn γ, by BD (1 mentions)
Ficoll paque plus, by GE Healthcare (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
12 protocols using facscan machine
1
Analyzing Brain Endothelial Cell Expression
2
Flow Cytometry Data Analysis
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Cells were analyzed on a FACScan machine (Becton Dickinson). Analysis was performed with FlowJo software (version 8-1.8.6, Treestar, Inc.).
3
Stable Transduction of Huh7.5.1 Cells
4
Cell Cycle Analysis by FACS
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A cell culture at OD600nm = 1 (1 ml) was harvested, resuspended with 1 ml 70% EtOH for fixation, and shaken at room temperature overnight. Fixed cells were suspended in 200 mM Tris, pH 7.8, with 0.4 mg/ml RNase and incubated for 4 h at 37°C, washed with FACS buffer (200 mM Tris, pH 7.8, 211 mM NaCl, 78 mM MgCl2), and subsequently resuspended in FACS buffer containing 0.05 mg/ml propidium iodide. Samples were sonicated and FACS analysis was performed with 1:20–1:10 dilutions of cell suspension in FACS buffer using a Becton Dickinson FACScan machine according to the manufacturers’ protocol. Fluorescence microscopy experiments were performed as described previously (Görner et al., 1998 (link), 2002 (link); Durchschlag et al., 2004 (link)).
5
Flow Cytometry Analysis of Stem Cell Markers
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To test cells for stem cell markers, 106 cells were suspended in 100 μl of PBS containing 1 μl of fluorescence-coupled antibody at room temperature for 1 hr in the dark. Anti-human-PE/FITC immunoglobulin isotype antibodies (BD Pharmingen) served as negative controls for gating. The cells were then washed twice, centrifuged, and subjected to FACS analysis using a FACScan machine (Becton Dickinson), and 10,000 living cells were analyzed (Koelling et al., 2009 (link)). For data evaluation, we used the WinMDlv2.9 program (Scripps Research Institute). The FACS Vantage SE (Becton Dickinson) was applied for cell selection and the Cell Quest Pro 2000 software package was used for analysis.
6
Cell Cycle Analysis by Flow Cytometry
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Flow cytometric analysis was performed with a FACScan machine (Becton Dickenson, Mountain View, CA). The data analysis and display were performed with the Cell-Fit software program (Becton Dickenson). Cell-Fit provides data from the flow cytometer and real-time statistical analysis of the data. After various treatments, cells (1 × 106/ml) were washed with 1 x PBS, fixed with 70% ethanol. Subsequently, cells were stained with propidium iodide (0.1 ug/ml) containing RNase at 1.5 ng/ml. The stained samples were kept at 4°C overnight before flow cytometric analysis for either measuring percentages of less G1 DNA contents of apoptotic cells or DNA profiles of cells in different phases of the cell cycle.
7
Whole-cell FACS for Cell Cycle Analysis
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Whole-cell FACS was carried out as described in detail in Sabatinos and Forsburg (2009 , 2015 ) and Sabatinos et al. (2015 (link)) with minor changes. To perform cell cycle analysis or microscopy, cells were fixed in 70% ethanol. Following rehydration in 50 mM sodium citrate, cells were treated with 1 µM SytoxGreen (Invitrogen, Carlsbad, CA) plus 10 µg/ml RNase A and incubated at 36°C for 1–2 h. Samples were then sonicated before being analyzed on a FACScan machine (BD Biosciences, San Jose, CA). DAPI staining was done by first rehydrating fixed cells in PBS and placing them to dry on positively charged glass slides. Mount solution (50% glycerol and 1 µg/ml DAPI) was then applied before cells were photographed on a Leica DMR wide-field epifluorescence microscope equipped with a 63×/1.62 NA Plan-Apo objective lens, 100-W Hg arc lamp for excitation, and a 12-bit Hamamatsu ORCA-100 CCD camera. Images were acquired using OpenLab version 3.1.7 (ImproVision, Lexington, MA) software and analyzed with ImageJ.
8
Cell Cycle Analysis via Flow Cytometry
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Cells were synchronized in the G0/G1 phase via incubating them in a serum-free medium for 24 hours, and then with DMEM and 10% FBS for 24 hours. Then the cells were trypsinized and washed three times with PBS. Afterwards the cells were fixed with 70% ethanol for 24 hours at -20°C.The samples were washed with PBS and dyed with PI/RNase staining buffer (BD Biosciences) for 15 minutes. Cell cycle analysis was performed via using fluorescence flow cytometry on a FACScan machine (BD Biosciences, San Jose,CA, USA).
9
Cell Cycle Analysis and Apoptosis Assay
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For cell cycle analysis, cells near 50% confluence were synchronized in the G0/G1 phase by overnight incubation in serum-free medium. Cells were then incubated in the complete medium containing 25 nM or 50 nM OTSSP167. After 24 hours (BGC823) or 18 hours (SGC7901) of incubation, the cells were trypsinized, washed with PBS, and fixed with 70% ethanol for 16 hours at −20°C. The samples were washed with PBS and stained with PI/RNase Staining Buffer (BD Biosciences) for 15 minutes. Cell cycle analysis was performed by fluorescence flow cytometry on a FACScan machine (BD Biosciences). For apoptotic analysis, cells were stained using an Annexin V/PI double staining kit (DOJINDO, Japan) according to the manufacturer's protocol.
10
Investigating Cinnamon Extract Cytotoxicity in K562 Cells
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K562 cells (1 x 10 6 cells/mL) in 25 mL medium with 10% (v/v) FBS were cultured in flasks. After incubating for 24 h, the medium was removed and replaced with medium containing cinnamon extract A, B, and C at concentrations of 50 and 75 µM. After incubating for 72 h, K562 cells (1 x 10 6 cells/mL) were collected, centrifuged at 400 g, and washed twice with PBS. They were resuspended in 0.5 mL of PBS with 50% ethanol by the addition of 0.5 mL of ethanol dropwise. After incubating at 4°C for 15 min, the cells were centrifuged and the ethanol was decanted. The cells were resuspended in 300 µL of PBS with 100 mg/mL of RNase A and 50 mg/mL of propidium iodide for 20 min. At 4°C, the cells were analyzed using a FACScan machine (BD FACSArial II, USA). The data were processed using MultiCycle software (BD FACSDiva software, USA).
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