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Cyan analyzer

Manufactured by Beckman Coulter
Sourced in United States

The CyAn analyzer is a flow cytometry instrument designed for high-performance cell analysis. It is capable of detecting and measuring multiple parameters of individual cells or particles in a sample. The CyAn analyzer uses light scattering and fluorescence techniques to provide quantitative data on size, granularity, and the presence of specific cellular markers.

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6 protocols using cyan analyzer

1

Intracellular Protein Staining in ESCs

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Intracellular staining was performed using BD transcription factors staining kit (BD), according to the manufacturer instructions with modifications. In brief, cells (1 × 106/tube) were incubated with BD Horizon fixable viability dye450 (1:1000) diluted in PBS (4 °C, 25 min). Cells were rinsed in PBS, resuspended in fixation buffer (4 °C, 45 min), and washed with ice-cold permeabilization/washing buffer. Fixed cells were incubated with primary antibodies (see supplementary table 1), diluted in permeabilization/washing buffer, overnight at 4 °C. The following day, cells were washed in permeabilization/washing buffer and incubated in secondary antibody (anti mouse-APC,1:500, Santa Cruz), for 45 minutes. After 3x washes in permeabilization buffer, cells were resuspended in ice-cold 500ul running flow buffer consisting of 1% bovine serum albumin (BSA) (Sigma-Aldrich) and 0.5 mM EDTA (Sigma-Aldrich) diluted in PBS. Flow cytometry analysis was performed using Beckman-Coulter CyAn analyzer (Beckman-Coulter), Summit 4.3 software (Beckman-Coulter) and Flowlogic software (Inivai technologies). Single cells were gated based on forward-side scatter profiles and dead cells excluded using violet Horizon viability dye (data not shown). Undifferentiated ESCs were used as a negative control to set gates.
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2

SSEA-1 Expression Analysis by Flow Cytometry

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Cells were trypsinized, washed once in PBS, and resuspended in PBS; dead cells were stained using the LIVE/DEAD Fixable Dead Cell Stain Kit (Invitrogen) according to the manufacturer’s instructions. Next, cells were fixed in 2% paraformaldehyde (PFA) for 15 min at room temperature, washed with PBS and stained with anti-SSEA-1 mouse monoclonal IgM antibody (mc-480, University of Iowa, Developmental Studies Hybridoma Bank, 1:400) at 37°C for 20 min. After a PBS wash, the cells were labeled with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgM (Millipore, 1:200) for 15 min at 37°C. After one PBS wash, stained cells were resuspended in PBS containing 1% FBS and 3% BSA, and at least 10,000 events of healthy cells were analyzed with a CyAN Analyzer (Beckman Coulter) and FlowJo ver.10 software (Tree Star, Ashland, USA). Control cells were not treated with primary antibody.
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3

Axl Expression Analysis in Lung Cancer

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Human small cell lung cancer A549 cell line and
nonsmall cell lung cancer cell line NCI-H249, obtained from American
Type Culture Collection (Manassas, VA), were grown in RPMI-1640 supplemented
with 10% fetal bovine serum. For FACS analysis, adherently grown A549
cells were detached by applying nonenzymatical citric saline buffer.21 (link) NCI-H249 cells were grown in suspension. To
minimize nonspecific uptake, the procedures were performed on ice.
Aliquots of cells were blocked with 10% normal goat serum, incubated
with anti-Axl primary antibody h173 (kindly provided by Vasgene Therapeutics
Inc., Los Angeles, CA) produced using composite human antibody technology20 (link) and goat antihuman Alexa Fluor 488 (Invitrogen,
Paisley, Scotland) in sequence. Subsequently, cells were measured
by flow cytometry (CyAn analyzer, Beckman Coulter). Cells without
the incubation of primary antibody were used as negative controls.
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4

Efficient DNA Repair Evaluation via Transfection

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MMEJ and SD-MMEJ plasmids were digested with the appropriate restriction enzymes (New England Biolabs) for 5 h and purified by ethanol precipitation. Aliquots were analyzed by gel electrophoresis to confirm complete digestion. A total of 2 × 105 CHO cells were seeded into each well of a 12 well plate (TPP) in 1 ml of complete medium. On the following day each well was co-transfected with 900 ng of linearized GFP reporter plasmid and with 100 ng of undigested pGL3-CMV-dsRed construct to normalize for transfection efficiency using Fugene 6, according to manufacturer's instructions (Promega). The pSV40-GFP vector (pGFP) was transfected in parallel as a positive control of GFP expression. Expression of GFP and dsRed was monitored by fluorescence microscopy (Carl Zeiss Microscope Axio Observer.A1) and flow cytometry. For flow cytometry, cells were harvested 24 h following transfection and resuspended in 0.5 ml of PBS with 2% FBS (Gibco, Invitrogen). Data were acquired using the CyAn analyzer (Beckman Coulter) and analyzed using the FlowJo software (Tree Star). GFP repair efficiency was calculated as the ratio of GFP-positive cells over the number of dsRed-positive cells.
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5

Cell Cycle and Death Analysis of Colon Cancer Cells Treated with Robenidine

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CT26 cells were seeded in a 6-well plate at 3 × 105 cells per well, incubated with increasing concentrations of RB (0, 100, 200, and 300 μM) for 24 h, and processed for cell cycle analysis. For cell death evaluation, both murine and human colon cancer cells were treated with RB for 1, 4, 8, and 16 h, after which both floating and attached cells were collected for further analysis.
Secondary to spectral overlap of RB with propidium iodide, DAPI was used to stain DNA and label dead cells. For cell cycle analysis, harvested cells were fixed with 80% ethanol on ice for 30 min and then incubated with 1 ml of DNA staining solution (1 μg/ml DAPI, 0.1% NP-40 in PBS) at room temperature for 30 min. DNA content was determined with FACS on a CyAn analyzer (Beckman Coulter, Brea, CA, USA) and analyzed with the FlowJo software (Ashland, OR, USA).
Cell death was determined with an Annexin V-Biotin Apoptosis Detection Kit. Briefly, cells were first stained with biotin-conjugated Annexin V in 1x binding buffer at room temperature for 15 min and then incubated with streptavidin-APC for 30 min. After being washed with PBS, cells were resuspended in PBS containing DAPI (0.2 μg/ml) for 5 min followed by FACS analysis. Cell death profile was analyzed with the FlowJo software. Necrotic cells were defined as DAPI+ and DAPI+/Annexin V+, and apoptotic cells were defined as Annexin V+ only.46 (link)
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6

FACS Analysis of Adherent and Suspension Cells

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For FACS analysis, the attached A549 cells were detached by incubation with citric saline buffer (0.135-M potassium chloride and 0.015-M sodium citrate) as described previously [24 (link)]. NCI-H249 cells grow as floating aggregates. Harvested A549 and NCI-H249 cells were aliquoted to 1×106 cells/tube and blocked with 10 % normal goat serum on ice. Cells were then incubated with 10 μg/ml hIgG-FAM or h173-FAM, respectively, for 30 min on ice. Subsequently, cells were washed twice with cold phosphate-buffered saline (PBS) and stained with 100-μl 4′-6-diamidino-2-phenylindole (DAPI, 1 μg/ml) diluted in PBS. For the quantification of fluorescence by flow cytometry (CyAn analyzer, Beckman Coulter), 10,000 viable cells (DAPI negative) were counted and analyzed. Each sample was repeated in triplicate.
For fluorescence microscopy analysis, A549 cells were planted on a BD Falcon 4-well chamber slide at 5×104 cells/well for 24 h. Cells were incubated with 300-μl complete culture medium containing 10 μg/ml hIgG-FAM or h173-FAM, respectively, for 0.5 h at 37 °C. After incubation, cells were washed with PBS and fixed in 4 % paraformaldehyde for 20 min at room temperature. Images were then acquired after mounting.
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