Plan your experiment well and choose your fluorescent antibodies in agreement with the optical configuration of the intended sorter. Note that our sorters may slightly vary in the optical configuration!
Stain cells according to your protocol. Unstained / non transfected cells are usually very helpful in estimating background signal. Further, consider the use of FMO controls and compensation samples depending on your panel requirements.
Please aim for a final cell concentration of
~5 x 106 cells / ml for a single cell multi-well plate sort
For a bulk sort provide cells with following concentrations depending on the used nozzle:
70 µm nozzle ⇒ 40 x 106 cells / ml
85 µm nozzle ⇒ 20 x 106 cells / ml
100 µm nozzle ⇒ 10 x 106 cells / ml
The minimum sample volume for cell sorting should exceed 200 µl. We can load samples in 1.5 ml eppendorfs, 5 ml FACS tubes or 15 ml falcons.
A maximum of 2% FCS / BSA is recommended in the suspension medium of cells to be sorted because otherwise cells become sticky and the optical property of the stream start to change significantly. Try to avoid phenol-red in case you want to bring your cells in cell culture medium.
If your cells have a tendency to form aggregates... then consider one of the following modifications:
► use calcium/magnesium free sorting buffer or add EDTA (< 5 mM)
► if the cell preparation induces increased cell lysis use 25 µg/ml DNAse I + 5 mM MgCl2 (no EDTA!)
► use 1% Accutase in sorting buffer
All samples need to be filtered directly before sorting (bring sufficient tubes with cell strainer caps ⇒ Falcon #352235).
Prepare collection tubes and add sufficient collection medium with regard to the expected yield / target cell number. You can calculate with following drop volumes/sort event assuming the "4-Way Purity" sort mask is used.
70 µm nozzle ⇒ ~1 nl
85 µm nozzle ⇒ ~2.2 nl
100 µm nozzle ⇒ ~2.5 nl
130 µm nozzle ⇒ ~6 nl
The choice of collection medium depends on the future application and is variable. The only exception is made for harmful volatile substances like Trizol or 2-Mercaptoethanol, which are not allowed as collection medium in our facility.
Following standard coolable collection containers can be used in parallel at all our locations.
4 x 1.5 ml eppendorf tubes
4 x 5 ml FACS tubes
2 x 15 ml falcons
6,12, 24, 48, 96, 384-well plates
At our Irchel sorter we additionally offer custom made coolable collection holders with following specifications.
4 x 2 ml eppendof tubes
1 x 5 ml FACS tube and 3 x 1.5 / 2 ml eppendorfs
1 x 15 ml falcon and 3 x 5 ml FACS tubes
1 x 50 ml falcon and 2 x 5 ml FACS tubes