Bookmark with More

Department of Pathology, University of Cambridge, UK

Visit the website

Pathology in Cambridge was one of the first departments in the UK to acquire a flow cytometer capable of separating cells when Alan Munro purchased an Ortho 50-H in 1979. The department has maintained a flow cytometry facility since that time and has thereby acquired extensive experience of operating a range of machines and managing a flow cytometry facility. We currently have a DakoCytomation MoFlo MLS high-speed cell sorter, a BectonDickinson FACScan analyser, a DakoCytomation Cyan ADP MLE analyser and Cytek DxP8 analyser.

The Flow Cytometry facility is located in the main Pathology building on Tennis Court Road. We are on the 3rd floor in rooms 331,333 and 334.

Who are we?

Nigel Miller click here to email or phone (7)65666

Nigel Miller is the full-time operator for the Flow Cytometry facility. Nigel has 27 years experience in flow cytometry. He provides a variety of services: on-hand troubleshooting and routine maintenance for all the machines, hands-on training for new users as well as running a course in Flow cytometry for the Graduate School which includes theory and practical elements, technical advice on machine setup and data analysis, in addition he operates the MoFlo sorter.

Nick Holmes email Nick or phone (3)33871

Nick is a senior member of Academic Staff. Nick oversees the financial management of the facility, and provides theoretical and practical advice in experimental design and data interpretation. He has 30 years experience in flow cytometry.

Training courses

Nigel Miller runs courses in the theory and practice of flow cytometry for the Graduate School. You can find out more about them here.

Analysers

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.