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Enquiries contact: westmead.cytometry@sydney.edu.au

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Objective

This document describes the safe operation of the CytoFLEX flow cytometer located at the Westmead Research Hub Flow Cytometry Core Facility (WRHFlow) in the Westmead Institute for Medical Research (WIMR). This document includes starting up the system, basic operation, cleaning, and shutting down the instruments when necessary by both staff and researchers. The procedures apply to users operating the CytoFLEX in room J.2.06, level 2 of WIMR. All personnel require training prior to independent operation of the instruments. All instrument operation is to be conducted by trained operators. Any assay to be run on the analysers involving hazardous chemicals must have appropriate approval.

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1. Turn on the CytoFLEX from the power switch located on the back of the instrument.
2. Log into windows if necessary. The all users account is password is cytoflex
3. CytExpert software and Westmead Cytometry confluence page should start automatically.
4. Click QC > start QC.
5. Initialise the instrument
6. Prepare QC beads if necessary. 1 ~1 drop in ~10 drops of H20.
7. Choose the correct configuration/bead lot number and click start QC.
8. Exit the QC (file > close QC) and prepare the workspace for default cell counting by loading an appropriate workspace.

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*All data collected will be synced to the WIMR server. If you would like to copy your data elsewhere copy it from this location. Your export folder can be easily accessed from the Scientific Platforms folder under My computer. Your data should be able to be accessed from the Scientific Platforms networked drive within WIMR, and via a username/password for non-WIMR users if desired (email wrhflow@sydney.edu.au to setup).

*Data should follow and be stored in the below listed format/location.

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1. Log in to PPMS screen locker. 
2. CytExpert should already be open (if it isn't start the software).
3. Click new experiment from template.
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4. Navigate to the template folder and choose a template.
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5. Make a new experiment in the CytExpert Data Folder using the provided syntax.
6. Templates are set to record 30 seconds of sample @30uL/min. this means 15uL of sample is used. These may be changed to suit your experiment .
7. Click 'Run' to start acquisition. Adjust the detector sensitivities if needed using the hand tool. 
8. Click 'Record' to record 15uL of sample (alternatively the 'Run' data is saved and can be used also).
9. Adjust the gate to exclude debri. New gates can be drawn if needed. 
10. Extract the events per uL statistic from the template. 
11. If desired export the PDF - final workflow to be determined. 
12. Save your experiment.
13. Log out of PPMS.

*the instrument is not shutdown by users. The instrument will enter a standby state after 10 minutes of inactivity

Cleaning the instrument

1. Clean the fluidics on the instrument by running the machine with 3 min 10% bleach, 3 min 5% decon & 3 min H2O on high. Leave the H2O tube on the instrument and put the instrument back to standby.
2. Close the software.
3. Check to see whether you are the last user for the day. If you are the last user for the day turn off the instrument. For CantoII, if you are the last user of the week perform a fluidics shutdown.
4. Log out of the instrument PC, complete the user survey.
   
   
5. Empty the flow cytometer waste container. Add 100ml of bleach into waste tank and refill the sheath. Bleached waste is poured down the sink gently under a running tap, minimising splashing. All other laboratory biohazardous waste is to be removed via the biohazardous waste disposal bins located in the lab.
6. Clean up and decontaminate the area with 70% ethanol.
7. Turn off the instrument using the green power button if you are the last user for the day.
   

   Data management

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