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Date of revision and approval
WIMR Document reference: WIMR-SWP-OP-WS-508.01
Date of approval: 6th 9th November 2018
Approval authority: Scientific Platforms Manager, Workplace Health and Safety Manager, Advanced Specialist Flow Cytometry
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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 (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. 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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- Acquisition of samples on the CytoFLEX must only occur after the approval of an associated project in PPMS.
- 100mL of 12.5% sodium hypochlorite (undiluted from the provided containers) must be added to the emptied waste containers daily and upon renewing the waste container.
- We recommend filtering samples prior to acquisition on the flow cytometry analysers to CytoFLEX to prevent sample acquisition issues arising from SIP clogging.
- Fixed samples may be filtered immediately before acquisition using the WRHFlow Westmead Cytometry provided 50um filters and plastic tweezers or tube top filters.
- Unfixed samples may be filtered before acquisition using the WRHFlow the Westmead Cytometry provided 50um filters and plastic tweezers or tube top filters in a biological safety cabinet.
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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@sydneywestmead.cytometry@sydney.edu.au to to setup).
*Data should follow and be stored in the below listed format/location.
- Log in to PPMS screen locker.
- CytExpert should already be open (if it isn't start the software).
- Click new experiment from template.
- Navigate to the template folder and choose a template.
- Make a new experiment in the CytExpert Data Folder using the provided syntax.
- Initialise the instrument by clicking 'Initialise' if needed (if needed the button will be activated).
- 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 30 40 seconds of sample @30uL/min. this means 15uL 20uL of sample is will be used. These may be changed to suit your experiment/adapted for different cell types.
- Click 'Run' to start acquisition. Adjust the detector sensitivities if needed using the hand tool.
- Click 'Record' to record 15uL 20uL of sample (alternatively the 'Run' data is saved and can be used also).
- Adjust the gate to exclude debridebris. New gates can be drawn if needed.
- Extract the events per uL statistic from the template.
- If desired export the PDF - final workflow to be determined.
- Save your experiment.
- Log out of PPMS.
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- Under no circumstances should there be any patient/person identiable data recorded on the instruments as the data is not privately held and is accessible to all researchers.
- User data is deleted after 7 days off the instrument computers, it is the responsibility of the user to ensure data has been transferred to a secure location.
- If you would like your data accessible from outside of WIMR, a user specific share can be setup. This works by creating a copy of a specified folder on university provisioned storage that is shared with the user. This requires an email to wrhflow@sydney to Westmead.Cytometry@sydney.edu.au and it is the users responsibility that all ethics approvals are complied with.
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- Filtering your sample beforehand can prevent clumps and erroneous data.
- Violet SSC provides better resolution for smaller particles and hence many templates trigger off violet SSC.many templates trigger off violet SSC.
- Abort rate should be kept below 2.5% for accurate cell counting.
Additional Information
Clean Up and Waste Disposal Requirements
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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:
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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