Cell sorting preparations
Our Institute currently hosts two FACS machines (BDFACSAria2 and BDFACSFusion) and one flow cytometer (Accuri C6) which are accessible for all members of the Institute and which can be booked via the internal booking system (www.molgen.mpg.de/Booking-Service). Every entry must contain the user's name, email address, a phone number and ideally a short experiment description. For FACS bookings, please also indicate whether you want to sort or only analyze your samples.
Prior to using the instruments for the first time or with a new project, it is advisable to contact us for planning your FACS experiments. When planning and designing FACS experiments, it is strongly recommended to check the spectral properties as well as potential spectral overlaps of the desired fluorophores or fluorescent proteins (e.g. using www.chroma.com/spectra-viewer, www.aatbio.com/spectrum/ or any other spectrum viewer).
There is no universal workflow for sample preparation that we can recommend, as projects seen in our facility are too versatile to pin it down to one protocol. Protocols for specific applications can be searched for at Current Protocols. However, all samples must be single cell suspensions and filtered (30- 70µm cell strainer) prior to analysis. As we are an S1 lab, only S1 samples can be analysed or sorted. The complete user rules can be found on the internal twiki.
To start with, general recommendations are:
- Read the user rules and stick to them please.
- The minimum sample volume for cell sorting should exceed 100 µL. We can load samples in 1.5 mL or 2 mL micro tubes (lids need to be cut off), 5 mL FACS tubes or 15 mL falcons.
- Unstained / non transfected/ single transfected cells are usually very helpful in estimating background signal. Additionally, for more complex experiments, consider the use of FMO controls and compensation samples (single stained samples) depending on your panel requirements. In case of doubt, please consult us.
- Depending on the used nozzle size, good cell concentrations to start with are:
70 µm nozzle ⇒ 30 x 106 cells / mL
85 µm nozzle ⇒ 10-20 x 106 cells / mL (our standard nozzle)
100 µm nozzle ⇒ 10 x 106 cells / mL
Please always bring buffer to be able to dilute the suspension further if required.
- A maximum of 2% FCS / BSA etc. is recommended in the suspension medium of cells to be sorted because otherwise the properties of the stream start to change significantly so that sorting becomes impossible. Please avoid phenol-red in case you want to bring your cells in cell culture medium.
- If your cells have a tendency to form aggregates, you may consider one of the following modifications:
⇒ use calcium/magnesium free buffer for your cells
⇒ add EDTA (2 - 5 mM)
⇒ add 25 µg/ml DNAse I + 5 mM MgCl2 (no EDTA then), if the cell preparation induces increased cell lysis
⇒ add 1% Accutase in sorting buffer
- Collection vessels and medium you should bring, if you want cells sorted:
⇒ You can collect your cells/particles into:
4x 1.5 mL/2 mL micro tubes
4x 5mL FACS tubes
2x 15 mL Falcon tubes
into one plate (6/12/24/96/384 well)
⇒ Prepare collection tubes/plates with sufficient collection medium with regard to the expected yield. Drop volumes per sort event, assuming the "Better Streams" sort mask is used, are:
70 µm nozzle ⇒ ~1 nL (i.e. 1 x 106 cells result in ~1 mL volume on top of your collection medium)
85 µm nozzle ⇒ ~2 nL
100 µm nozzle ⇒ ~2.5 nL
130 µm nozzle ⇒ ~6 nL
⇒ Collection medium depends on your downstream application. Harmful volatile substances like Trizol or 2-Mercaptoethanol are not allowed as collection medium in our facility.
Our reward will be in a paper‘s acknowledgement or co-authorship depending on the extent of service and input from our side. Any helpful intellectual input from our scientific members into the project during planning, execution and analysis of the experiments justifies a co-authorship on a manuscript that contains data generated in our facility.