Date of revision and approval

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Enquiries contact name: Xin Wang, Suat Dervish, Edwin Lau

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

Objective

This document describes the safe operation of the CytoFLEX flow cytometer located at the Westmead Research Hub Flow Cytometry Core Facility 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. OGTR requirements for safe work in a PC2 laboratory apply.

Hours of operation and emergency contacts

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Advanced Specialist on 0-8627 3216

Cytometry/Imaging/EM Manager on 0-8627 1820

Scientific Platforms Manager platforms Director on 0-8627 3210

Email: westmead.cytometry@sydney.edu.au

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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.
   
4. Navigate to the template folder and choose a template.
   
5. Make a new experiment in the CytExpert Data Folder using the provided syntax.
6. Initialise the instrument by clicking 'Initialise' if needed (if needed the button will be activated). 
7. Provide 50uL (minumum) of sample to be counted (with or without facility provided DAPI - 5uL to can be added is used). Samples can be provided in eppendorf tubes or 5mL FACS tubes. Templates are set to record 40 seconds of sample @30uL/min. this means 20uL of sample will be used. These may be changed to suit your experiment/adapted for different cell types.
8. Click 'Run' to start acquisition. Adjust the detector sensitivities if needed using the hand tool. 
9. Click 'Record' to record 20uL of sample (alternatively the 'Run' data is saved and can be used also).
10. Adjust the gate to exclude debris. New gates can be drawn if needed. 
11. Extract the events per uL statistic from the template. 
12. If desired export the PDF - final workflow to be determined. 
13. Save your experiment.
14. Log out of PPMS.

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Training is conducted by a super user or the scientific platforms staff with competency demonstration necessary before authorisation of access. Competency is assessed via completion of a competency quiz.

Emergency procedures

Emergency procedure folder can be found here in the sample prep lab:

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You can also access all these information via WIMR intranet:

https://intranet.westmeadinstitute.org.au/workingatwmi/HandS/Pages/Sarah%20Johnston.aspx

All emergencies need to be reported to the emergency contacts listed above. Specific chemical exposure procedures are below.

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