05_SWP_Cell Cycle Analysis with Flow Cytometry_JC

Safe Work Procedure
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School of Medical Sciences ORU/MDU
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The Writing Safe Work Procedures Guideline (OHS027) should be consulted to assist in the completion of this
Safe Work Procedure Title and basic description
Title: Cell Cycle analysis with flow cytometry
Description: To determine cell number and DNA content using flow cytometry
Associated risk assessment title and location: 05_RA_Cell Cycle analysis with flow cytometry_JC
Describe the activity or process
Harvest all the adherent and floating cells in the dish or flask. This means transferring into a
centrifuge tube, the medium that the cells have been growing in and the pre-trypsin wash, as well
as the trypsinised cells themselves.
Centrifuge at 300 rcf for 5 minutes. Resuspend the cells in 10mls of warm PBS, then take out an
aliquot to count while you centrifuge again at 1500rpm for 5mins. Resuspend the cells in cold PBS
at a concentration of 106/ml.
Transfer 250ul of cells (i.e. 2.5x105 cells) to a 1.5ml eppendorf tube. Add an equal volume (250ul)
of ice-cold 70% ethanol and mix by inversion several times.
Incubate tube on ice for at least one hour. At this stage the cells can be left for up to ten days at
Centrifuge the cells at 4oC at 1500rpm for 5 mins. Discard the supernatant and add the following
mix to the cells :
5ul Propidium Iodide (1mg/ml stock,
made up in water)
2ul RNase (0.5mg/ml)
475ul PBS (warm)
Mix gently by inversion.
Incubate tube in dark (PI is light-sensitive) at 37oC for 30 mins.
Transfer sample to FACS tube and run on flow cytometer at low speed (< 200 cells/sec). Specific
protocols associated with each flow cytometer (refer to flow cytometry facility).
List all resources required including plant,
chemicals, personal protective clothing and
equipment, etc
Propidium iodide
Flow Cytometer
Ethanol (70%)
FACS tubes
Eppendorf tube
Page 1 of 2
Safe Work Procedure
Date Effective: 01/01/2007
Uncontrolled document when printed
Current Version: 1.2, 15/08/2007
List potential hazards and risk controls including
specific precautions required
1) Toxic chemical risk - Propidium Iodide - Low Risk.
Mild irritants, potential mutagen. Risk Controls: use of protective clothing (lab coat, gloves and safety glasses), use of
disposable pipette tips
2) Electrical Hazard – Flow cytometer – Low Risk. Potential electrical hazard. Risk Controls: Equipment routinely
tested and tagged. User is appropriately trained.
3) Fire Hazard – Ethanol – Low risk. Risk Controls – keep confined, no ignition sources nearby.
List emergency shutdown instructions
Shutdown button located at entrance to lab.
Emergency contact Renee Szokolai ext 58497
List clean up and waste disposal requirements
Dispose of material in biological waste bin.
List legislation, standards and codes of practice used
in the development of the SWP
NSW OHS Act 2000
NSW OHS Regulation 2001
Code of Practice for the Labelling of Workplace Substances
AS/NZS 2243.2:2006. Safety in laboratories. Part 2: Chemical aspects
AS/NZS 2243.3: 2006 Safety in laboratories Part 3: Microbiological aspects and containment facilities
AS/NZS 2243.6-1990. Safety in laboratories. Part 6: Mechanical Aspects.
AS/NZS 2243.3:2006 Safety in Laboratories Part 7 Electrical aspects
AS/NZS 2161.1:2000 Occupational Protective Gloves – Selection, Use and Maintenance
Supervisory approval, training, and review
Plant custodian:
List competency required – qualifications, certificates, licencing, training - eg course or instruction:
Training as per training needs analysis, Induction to Lab, Training on this SWP
Page 2 of 2
Safe Work Procedure
Date Effective: 01/01/2007
Uncontrolled document when printed
Current Version: 1.2, 15/08/2007