Protocols and tools

Flow Cytometry Protocols Editors: Hawley, Teresa S., Hawley, Robert G. (Eds.)

Modern Concepts of Flow Cytometry Barbara Roth (editor)

Although workflow is pretty much similar to conventional Flow Cytometry is good to get acquainted with how to prepare for spectral Flow Cytometry

: The Power of Reagent Titration in Flow Cytometry

(Paper on Cytometry Part A Journal)

: See the configuration of our instruments and design your panels based on them.

• Only BSL1 cells; No infectious or pathogenic samples are allowed. All human samples from screened persons only. Transduced cells must be certified RCV negative. Bacterial and iPS samples must be discussed with personnel.
• To remove debris and excess antibody, wash samples in PBS or FACS buffer.
• Cell concentration depends quite a lot on your cells and experiment. With high efficiency and resolution experiments (e.g. Cell cycle, plate sorting and big or “sticky” cells) slower is better, 1 million/ml is usually a quite good concentration. With tube sorting for instance you can test 5-10 million/ml and have buffer on hand to dilute if needed.
• Remember that there should be around 100ul dead volume in the sample (amount in the end that you should not run to prevent air going into the system)
• If you use FACS buffer or PBS with low serum if any (max 2 % filtered). If necessary you can use medium but remember that high amount of serum and phenol red will give some fluorescence background.
• With tissue culture you might need to optimize all the steps based on the tissue in question. Tissue samples tend to give lot of fluorescence background from cell debris. Consider using viability dye.

Analysis

• For set up, it is always advisable to have an unstained control sample and multicolor experiments will need all separately stained controls for compensation.
• Multicolour experiments should be planned thoroughly to avoid unnecessary over-spills and compensation spread; Small populations with brighter colours and sub-populations should not have spilling from previous population…
• Remember to use Fc-blockers if needed/provided.
• Always prefer primary antibodies if possible.
• When fixing the samples please keep in mind the experiment in hand; What fixative do you need, do you need to study viability, staining first or after (intracellular or surface stainig, some antibodies might lose fluorescence), cells become buoyant so be careful with handling.

Sorting

• Always filter samples before sorting and remember to have tubes and buffer for the sort collection. You can pre-coat the inner walls of the collection tubes with a protein containing solution.
• For collecting few events-1000 cells, use plate. For thousands, up to few hundred thousand you can use 1-2ml tubes. About million cells into 5ml tube and several millions into 15ml tube.
• Consider using viability dye
• Also consider sample temperature; if the cells are standing for long periods of time cooling the sample and instrument (sorters) to 4°C might be a good idea. Plate sorting or fast live analysis might be enough to have in RT.