Cell Sorting
The effectiveness and quality of cell sorting is highly dependent on the sample being in a single cell suspension with minimal aggregation. In order to achieve this cells have to be treated with care to ensure that to minimise cell death.
What to bring to the sorter?
Please bring -
Labelled cells and any controls required (please discuss these with the sort operator). The samples must be filtered through a 50um mesh just before the sort. Filters are available from the facility. Additional sort buffer to dilute the sample if necessary.
Collection tubes and plates – please bring collection tubes and plates to the sort along with your samples. Collection tubes should contain a suitable liquid (media containing serum, PBS or lysis buffer) and be clearly labeled.
Preparation of adherent cells for cell sorting
Sort buffer
This is the buffer in which your cells will be in during the sorting period. The media can be either Ca/Mg-free PBS or the normal culture media used with the cells but must be without any added serum. .
The addition of 25mM Hepes to the media helps to maintain the pH. The addition of up to 2.5mM EDTA can help prevent cells aggregating.
Collection medium
This is the media we will use to collect the cells into. We can collect the cells into any media - PBS or a nucleic acid stabilising agent (eg RLT Buffer) can be used if the cells are to be used for downstream genomic techniques. We can collect into culture media or serum if the cells are to be grown onwards following sorting.
Cell concentration
The best concentration for the cells can vary depending on the cell type. A good place to start is to have the cells in the range 5 x 106 – 10 x 106 per ml. If you have less than 5 x 106 cells make the sample volume 0.5ml.
Sorting into multi-well plates
Cells can be sorted into 6-384 well plate formats. Cells can also be sorted onto slides.
Please bring multi-well plates with collection medium or culture medium in the wells, 50-100ul per well is sufficient for 96 well plates. Cells can also be sorted directly into lysis buffer if required.
*Please let the facility know if you require collection into multi-well plates ahead of time.