Yeast cultures in log-phase growth were diluted 10X in 50 mM sodium citrate buffer. Flow cytometry was performed with a CyAn ADP 9 Analyzer (#CY20130, Beckman Coulter, Inc.) using a 488 nm solid-state laser to excite the yEGFP fluorophore. Fluorescence emission was detected with a 530/40 filter. All Flow Cytometry Standard file formats (.fcs) were analyzed using MATLAB (The Mathworks, Natick, MA) with a custom FCS extraction and analysis script.
Cyan adp 9 analyzer
Manufactured by Beckman Coulter
Sourced in Canada
The CyAn ADP 9 Analyzer is a flow cytometry instrument designed for multi-parameter analysis of cells or particles. It is capable of detecting and measuring various physical and fluorescent characteristics of individual cells or particles within a sample. The CyAn ADP 9 Analyzer provides rapid, high-throughput data acquisition and advanced analysis capabilities.
Automatically generated - may contain errors
3 protocols using cyan adp 9 analyzer
1
Yeast Cell Fluorescence Quantification
2
Preserving E-cadherin in hESC Dissociation
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To preserve membrane E‐cadherin expression, hESCs were dissociated with Cell Dissociation Buffer (Life Technologies, Waltham, MA, http://www.lifetechnologies.com ) for 14 minutes, filtered through a 40‐μm cell strainer (BD), and immunolabeled with either Alexa‐Flour647 mouse anti‐human E‐cadherin antibody or isotype control (BD Biosciences, Mississauga, Canada, http://www.bdbiosciences.com ) for 120 minutes on ice. Live cells identified by 7‐AAD exclusion were analyzed for E‐cadherin and TCF‐eGFP expression on a CyAn ADP 9 Analyzer (Beckman Coulter, Mississauga, Canada, https://www.beckmancoulter.com ). Detailed procedures are described in Supporting Information.
3
Yeast Fluorescence Quantification via Flow Cytometry
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Yeast cultures were diluted 1 in 10 in 50 mM sodium citrate buffer prior to being sampled by the flow cytometer. An IntelliCyt HyperCyt autosampler attached to a Beckman Coulter CyAn ADP9 analyzer was used for the collection of all flow cytometry data. Yeast cells were gated with a small forward and side scatter gate (~40% of the population) to reduce variability from extrinsic sources. A 488 nM laser and a 530/40 band-pass filter were used to excite and detect yeGFP fluorescence. FCS files were analyzed by custom scripts created in MATLAB R2013a (Mathworks Inc, Natick, Massachusetts). Mean fluorescent intensity values and standard error values were calculated from this data.
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