Sample preparation for cytometry

General

Plan your experiment well and choose your fluorescent antibodies/proteins in agreement with the optical configuration of the intended cytometer. The cytometers can vary significantly in their optical configuration!

For complex fluorescent panels we highly recommend to test the experiment on an analyzer before booking an operator-based sort in order to avoid trouble during your sorting session.

Sample preparation

Your cells must be in a single cell suspension when loading to the cytometer.

Samples can only be loaded in 5 ml FACS tubes made of polystyrene (clear plastic e.g. FAL352058) to the analyzer (sorters are more flexible as mentioned below).  Polypropylene tubes (milky plastic like e.g. falcons or Eppis) will not pressurize. Minimum sample volume is 200 ul.
If you have sample volumes < 200 ul you can place an additional micronic tube in the 5 ml tube (e.g. Vitaris, Product no.: 32022-MIC).

 Following aspects will improve your sample recording at the cytometer.

  1. The sample buffer should not exceed a concentration of  2% FCS / BSA because many cell types start forming aggregates at higher concentration. Use phenol-red free medium in case you want to bring your cells in your culture medium (min 2% serum). A basic sorting buffer consists of 1xPBS, 1mM EDTA, 25mM HEPES (pH 7.0), 1-2% serum or BSA.
  2. Filter all samples before loading (mesh size 30-50 µm, e.g. cell strainer caps  ⇒ Falcon #352235). If your cells quickly form aggregates you should filter them directly before loading to the instrument.
  3. Dead cells increase soluble DNA in the sample which can lead to severe clumping. Try to optimize the cell preparation and/or add 25 µg/ml DNAse I + 5 mM MgCl2 (no EDTA!).
  4. If your cells keep on forming aggregates... you may consider some of the following modifications:

► increase EDTA concentration up to 5 mM (cations promote cellular adherence)
► Use 1% Accutase in sample buffer
► Use FCS dialyzed against Ca/Mg free PBS

 

Stain cells according to your protocol. Additionally, prepare all relevant control samples for your experiment which could be for instance:

► Unstained and/or empty-vector transfected cells for estimating background signal

► Single stained control samples if your panel requires compensation

► L/D marker positive control

► Consider the use of FMO controls for evidence based gating decisions

 

 

Sample concentration for cell sorting

We can load samples in 1.5 ml tubes, 5 ml FACS tubes or 15 ml falcons to the sorter. The minimum sample volume for cell sorting should exceed 200 µl.

The time requirements for your sort can be optimized by sticking to following cell concentrations in your sample

~1 - 5 x 106 cells / ml for a single cell multi-well plate sort (depending on the frequency of positive cells).

For a bulk sort you should aim to reach 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
130 µm nozzle   ⇒   3 x 106 cells / ml

 

Collection of sorted cells

Prepare collection tubes and add sufficient collection medium with regard to the expected yield / target cell number. You can calculate with following droplet volumes per sort event assuming the standard "4-Way Purity" sort mask is applied. 

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 (except for harmful volatile substances like Trizol or 2-Mercaptoethanol which are prohibited in our facility as collection medium) . Following points may be considered:

  • Protein is important in collection buffer! Fragile cells often benefit from being sorted into 100% serum used as collection medium
  • Avoid mismatch of collection buffer and sheath. The calcium chloride in most culture medium is not compatible with the phosphate component of the sheath which can lead to precipitation of calcium phosphate crystals and subsequent cell damage => collect cells in PBS + sufficient serum quantities instead of culture medium
  • Many cell lines show better post-sorting performance when collection medium is hold at room temperature instead of 4°C (not relevant for PBMCs)
  • Cells which do not end up in collection medium after sorting have a high chance of dying because of buffer evaporation. Therefore, always add sufficient amounts of collection medium and choose collection container that fit to the number of sorted cells (collecting 5000 cells in a 15 ml falcon is obviously a bad idea)

Following standard coolable collection holders allow parallel sorting in either...

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

Troubleshooting single cell sorts for clonal populations

The sorter can reliably sort single drops in each well of a multi-well plate. The survival rate of the sorted cells can range from 0-99% and depends on the preparation as well as the characteristics of the cells. Some cells are very sensitive to the pressure changes that occur during the sort process, while others require cell-to-cell contact for proliferation and will not expand in the culture vessel.

  • We recommend to use a large diameter nozzle of 100 or even 130 µm to reduce sorting induced stress.
  • Prepare well plates in advance and allow medium to equilibrate for temperature and CO2. Use the cell incubator provided by the facility to store your plates and return them immediately after sorting.
  • Some cells strongly benefit from using preconditioned medium in the collection wells. Make sure you have performed a high speed centrifugation step to remove floating cells and debris before filling collection plates.
  • Some users reported improved survival rates when ROCK inhibitor (e.g. 10 µM Y-27632 for 24h post sort) was added to the culture medium. Please check literature for your cell type and suggested concentration of inhibitors.
  • As a last attempt one can try to sort a single cell on a layer of feeder cells. Feeder cells can be of the same type of your target cells or a different cell type. They are usually mitotically inactivated by radiation or chemically (e.g. mitomycin C). After sufficient proliferation of your target cell one may run a second bulk sort to remove remaining feeder cells.