The information that you provide in the request file will allow us to choose the best nozzle size, pressure and optical component in order to get the best results out of the sort. For this reason, we would like to ask you to complete the request form (here is the online form Sort Request) before the sorting precisely. Please let us know if you find any problem in your sorted cells (low viability, very low cell number, contamination…). It will help a lot to improve the quality of our sorts.
Successful sorting depends entirely on the quality of the input sample. Please bring your cells in FACS tubes in a buffer the cells are happiest in. Sample buffer should have a protein concentration of max 2% and content no Phenol Red.
There is no optimal concentration that would work for all cell types and different sort set-ups, but we recommend to start with the following concentrations:
- 20 – 40 million/ml for lymphocytes
- 10 - 15 million/ml for activated cells / smaller cell lines
- 5 -10 million/ml for larger adherent cells
- 1 million/ml for special applications (e.g. pancreatic cells)
Cell concentration above 4x107 per ml is not recommended. We might need to dilute the sample so please bring an extra buffer to the sort.
Count cells right before sorting, not before staining procedure, which often results in considerable cell loss. Always start staining with more cells than needed for the sort.
Please filter your sample just before starting the sorting to remove the aggregate. Rule of thumb is that the filter size should be around half that of the sort nozzle used. Therefore, cells that will be sorted through a 70 µm nozzle have to be filtered by 30 µm filter and cells through a 100 µm nozzle with 50 µm filter. Filtering cells greatly reduces the probability of clogging the instrument during sorting. As a general rule, we will not sort unfiltered samples. We want to ensure a successful sort and once the instrument is clogged, it may take as long as an hour to bring the instrument back to its original configuration.
We strongly recommend using viability dyes to exclude dead cells. For multicolor experiments, please provide FMOs (Fluorescence minus one) controls to facilitate gating.
We recommend that you collect your cells into a larger volume of the buffer. This avoids stressing the sorted sample but this has to be decided considering your aim. The larger the volume of collection buffer the lower the stress on the cell. But, this will mean you can collect fewer cells per tube.
Collect your cells into neat FBS/FCS or high concentration FBS/FCS cellular media. This can improve the survival of your sorted sample.
The number of cells you need to bring depends on:
- The number of the cells you require for downstream work.
- The frequency of your cell population in your pre-sorted sample.
- LMU Sort Request (16 KByte)