We have 3 analysers.
The BD FACScan is a single laser machine with 488 nm illumination and detects 5 parameters, 2 light scatter ('forward' or low-angle scatter and 'side' or 90-degree scatter) and 3 fluorescence bands (515-545 nm, 564-606 nm, 650+ nm). Typically this machine is used to detect emission from FITC, PE, PE-CY5 or PerCP, but other fluorochromes may also be used.
The Cyan ADP MLE provides 3 colour excitation at 351nm, 488nm and 635nm with a maximum of 9 fluorescence and 2 scatter parameters; it can analyse at rates of up to 50,000 events per second. This cytometer is useful for those searching for rare cells, those with applications requiring more than 3 colors to define a cell population or wishing to use a fluorochrome requiring UV or red light excitation. Recommended fluorochromes for the CyAn include but are not limited to: FITC, PE, PE-Tx-Red, PE-Cy5, PE-Cy7, Alexa-Fluor 350, DAPI, Hoechst 33342, Indo-1, APC, Alexa-647 and APC-Cy7. The CyAn is designed to be user friendly and most users are comfortable with it after one or more training sessions from Nigel.
NEW The Cytek DxP8 provides 3 colour excitation at 488nm, 561nm and 637nm with a maximum of 8 fluorescence and 2 scatter parameters. This cytometer is useful for anyone using one of the many flavours of red fluorescent protein (mRFP1, mCherry, dTomato etc.). It will also help those with applications requiring more than 3 colors to define a cell population or wishing to use a fluorochrome requiring red light excitation, particularly those who have large cells or samples with small aggregates (which cause a problem for the Cyan's small bore tubing) . Recommended fluorochromes for the Cytek include but are not limited to: FITC, PE, PerCP-Cy5.5, PE-Cy7,GFP, RFPs, Texas Red, APC, Alexa-660 and APC-eFluor780. The Cytek is based on FACScan fluidics and is designed to be user friendly and we expect most users will comfortable with it after one training session from Nigel.
Here are some suggestions for 5, 6 and 7 colour fluorochrome combinations which should work well on the Cytek DxP8 Also note that the data files (FCS3) from the DxP8 can only be opened in FlowJo at present.
The machines are user-operated but you need authorisation and an account from Nigel or Nick. Training will be provided as needed.
The cell sorter
Cell separation needs are provided by a DakoCytomation (now BeckmanCoulter) MoFlo MLS high-speeed cell sorter. This machine can handle total input rates of 35,000 cells per second (1.2x10e+8/hr). It can sort cells in bulk or deposit any chosen number in multiwell trays of 384 - 6 wells or onto slides. It takes about 2 minutes to 'clone' cells into a 96-well tray (assuming desired population is about 1% of input mixture). The machine can sort 4 different populations simultaneously. maximising the use of your samples. Our MoFlo has 3 lasers and 8 detection parameters. Two of the lasers are multiline so we can illuminate at more than 10 different wavelengths from UV (351 nm) to red (647 nm). Detection can be configured for many different fluorochromes, including fluorescent proteins (eGFP, EYFP, mRFP1, mCherry etc.). The machine can sort many particles other than mammalian cells e.g plant protoplasts, nuclei, microbeads, bacteria, protozoa and subcellular organelles. We welcome enquiries regarding possible uses. Only Nigel can operate this machine.
In order to cover the running costs of the machines we do have to make a charge for use. The three analysers are charged at £20/hr of use; this charge covers the cost of operation. The Moflo sorter is charged at £50/hr; this is only on a partial cost recovery basis and is at present being subsidised by the Division of Immunology and the Department of Pathology. Despite this, the use of all these machines is open to any users within the University. Over the past year we have helped about 70 users from at least 10 institutions, the majority from within the School of the Biological Sciences.
We also provide, free of charge, two stand-alone computer workstations for flow data analysis for users of the facility. The Mac G4 has BD Cellquest and FCS Press software. The PC runs Summit, FlowJo and Multicycle (a specialised application for cell-cyle analysis).