Flow Cytometry and Cellomics

The High Content Screening and Flow Cytometry group provides training and experimental set up advice for new users, in addition to cell sorting for the Unit Programmes and external collaborative projects. Help with Flow Cytometry data analysis is also provided.

Flow cytometry is a powerful tool for the analysis of all types of cells and microparticles.

The main applications using Flow Cytometry in the Toxicology Unit are:

  1. Multicolour phenotyping (we can measure up to 16 colours in an individual cell).
  2. DNA analysis and cell cycle kinetics.
  3. Cell death and apoptosis.
  4. Cell proliferation.
  5. Calcium flux measurements and other functional assays.

Recently it has been widely used to analyse and separate CRISPR transfected cells.

Flow cytometers may be divided into analysers and sorters. In addition to analysing a large number of cells very quickly, it is possible to recover cells identified by analytical flow cytometry for further study, such as re-growth, extraction of nucleic acids or additional functional studies.

We have four Flow Cytometers available in the core facility of our Unit:

  1. BD FACS Calibur (x2) – analyser. Software: CellQuest Pro v6.0, FlowJo.
  2. BD FACS Canto II – analyser. Software: Diva 8.0.1, FlowJo.

These two analysers are capable of detecting up to six fluorescent signals (FITC, PE, PE-Cy5.5, PE-Cy7, APC and APC-Cy7) with two lasers (blue argon and red HeNe).

3.  BD FACSAria II – cell sorter and analyser.

This has five lasers (Blue (488 nm), Yellow Green (561 nm), Red (640 nm), UV (355 nm) and Violet (405 nm)) and can detect up to 16 colours simultaneously. It also has an aerosol management installed.

The FACSAria can perform two- and four-way sorting as well as automatic cell deposition for sorting onto tubes, TC plates or slides. Sort rates are 20,000 cells per second for lymphocytes and 10,000 cells per second for cells >10um.

It is strongly recommended that the choice of dyes is discussed prior to ordering any antibodies/compounds to ensure they fit the instrument configurations. New users should contact Dr Lucia Piñon (lp54@le.ac.uk, tel: 0116 252 5219) for training, and experimental set up advice.  Once users are trained they operate the FACS analysers by themselves, whilst the FACS sorter (AriaII) is operated solely by the manager of the facility (Dr Lucia Piñon).

High-content Screening (HCS) uses automated microscopy, multi-parameter image processing, and visualization tools to extract quantitative data from adherent cell populations. HCS typically employs fluorescence imaging of samples in a high-throughput format and reports quantitatively on parameters such as spatial distribution of targets and individual cell and organelle morphology. It is a powerful tool for research into cellular and systems biology and drug discovery. Cell and nuclear masks are used for automated demarcation, robust functional probes for cell health interrogation, a flexible assay workflow for automated processing, and ample fluorophore choice for easy multiplexing.

Some examples of the bioapplications that can be performed are:

  1. Analysis of cell viability.
  2. Proliferation.
  3. Apoptosis.
  4. Cell cycle and DNA repair.
  5. Migration.
  6. Mitochondrial health.

In the Unit we have an ArrayScan (Cellomics VTI) with a live imaging mode (temperature and CO2 levels controlled).

 

The activation of Bax in MCF-7 cells by TRAIL

This is an example of a study performed in the Unit.
The activation of Bax in MCF-7 cells has been quantified using a specific antibody to activated Bax and a Cellomics VTI machine.

Selected publications

Enhancement of CD154/IL4 proliferation by the T follicular helper (Tfh) cytokine, IL21 and increased numbers of circulating cells resembling Tfh cells in chronic lymphocytic leukaemia.

Ahearne MJ, Willimott S, Piñon L, Kennedy DB, Miall F, Dyer MJ, Wagner SD.

Br J Haematol. 2013 May 27. doi: 10.1111/bjh.12401.

Different pathways lead to mitochondrial fragmentation during apoptotic and excitotoxic cell death in primary neurons.

Young KW, Piñon LG, Bampton ET, Nicotera P.

J Biochem Mol Toxicol. 2010 Sep-Oct;24(5):335-41.

Mitochondrial fragmentation and neuronal cell death in response to the Bcl-2/Bcl-x(L)/Bcl-w antagonist ABT-737.

Young KW, Piñón LG, Dhiraj D, Twiddy D, Macfarlane M, Hickman J, Nicotera P.

Neuropharmacology. 2010 Jun;58(8):1258-67. Epub 2010 Mar 20.

Flow Cytometry and Cellomics