FAQs concerning FACS
First time users and users with variations in experimental design or new sort approaches are advised to contact us beforehand.
Registration: First Time Users have to register online to get access to the scheduler(link is external).
Requested Forms: First Time Users have to fill out the First time user registration for billing puposes.
The users must send the completed Sort registration form with specifications to the staining used, appropriate BSL levels and GenTech safety via Email to cellsort.mih@mh.tum.de, where the confirmation is provided by the facility.
The sort facility provides service: Monday: 12 am - 4 pm, Tuesday to Thursday: 10 am - 4 pm, Friday: 10 am - 3 pm. Exceptions to these opening times cannot be granted. Sorting appointments exceeding 4 hours require clarification on the importance of the experiment and acceptance by the staff; the facility reserves the right to charge extra or disproportionate bookings accordingly. The facility will adjust the sort timings to the real sort duration, since the billing is calculated for every half hour.
Clients have to adhere to their booked time, as we cannot grant additional sort time due to delayed arrival. Short term cancellation (<12h) may be fully charged by the facility.
The final choice for the sort instrument used will solely be made by the operator.
Samples containing tracers or other radioactive substances cannot be sorted within our facility. We abide that no carcinogenic, evaporative, harmful and irritating buffers will be used by our clients. Users have to declare the source and all the sample related BioSafety measures in their sort registration.
No sorting into harmful evaporative substances like 2-Mercaptoethanol or Trizol will be done!
Our service will be charged in accordance with our “Concept and Terms of use”, which relies on suggestions and rules by the DFG concerning Core facilities.
All formal presentations or publications containing data from work performed at our Flow Cytometry Core Facility must be identified. For further information, see Rules for Acknowlegment and Collaboration.
The choice of instrument strongly correlates with your experiment.
First time users and users with variations in experimental design or new sort approaches can contact us beforehand to plan the experimental needs accordingly. The users are required to send us the completely filled sort registration form with specifications to the staining used, appropriate BSL levels and GenTech safety.
You can find the instrument setup for all available cell sorter online.
Sorting is performed with a 70µm nozzle (default setting) or 100µm nozzle depending on the cell sizeSpeedenrichment is a service user can book within our facility to enrich for small target cell populations as a substitute for MACS positive enrichment. For further information, the principle is described in detail below; To check if Speedenrichment is feasible for your experimental needs please clarify with a detailed experimental plan with the core facility staff beforehand.
MoFlo Astrios EQ system: six different populations into Eppendorf tubes or 5ml FACS tubes; two populations into 15ml or 50ml Falcon tubes, and only one population in a plate format.
FACS Aria III sorter: two different populations in 15 mL Falcon tubes; four populations into Eppendorf tubes or 5 mL FACS tubes at one time. Plate Sort yields only one population at a time, but up to four different populations can be programmed for this deflection site subsequently.
S1 sorter is considered semi-sterile as the instruments are not enclosed within a Cabinet, but sort chambers and sample holders are separated from the environment. Sorts on Astrios S2 can be done under cell culture sterile conditions with a BioSafety cabinet ensuring sterility. Regardless of wether sterile or semi-sterile, the sheath fluid within our sorters passes a 0.2 µm filter. Instruments are decontaminated daily with 5% bleach, detergent and dd-Millipore water regarding sample line and nozzle, while surfaces are wiped with 80% ethanol between users. Daily instruments start-up includes a longer sample run of 70% ethanol before QC measures. Weekly cleaning of exposed surfaces with 3.5% Desmozon ensures further GLP standards. Monthly, we control for sheath contamination by incubating sheath specimens on blood agar culture media at 37 °C.
After sorting, we recommend to culture cells with media containing pen/strep.
Sterile sample preparation can be handled at our facility, all work steps, even on the device on site can be done under a BioSafety cabinet.
Samples: All sorting materials / samples and controls must be pre-filtered through a 35 µm cell strainer before sorting. Filters (sterile/unsterile) are available at the sort facility. Cell strainer size can be adjusted for cell size and nozzle setup. Filtration of cells before the sort is mandatory!
Cell collection: Target cells should be sorted in suitable buffers (FACS Buffer; Cell culture media; PBS etc.). We recommend coating the inside walls of your collection tubes with the collection buffer of your choice or FCS (fetal calf serum).
Available sorting formats (Aria and Astrios) include 50ml and 15ml Falcon tubes; 5ml FACS tubes; 2ml and 1.5ml Eppendorf tubes; 6-; 12-; 48-; 96- and 384-well plates; (Astrios) 1536-well plate in addition.
We use unstained cells (which include live/dead marker) for setting up the instrument, single stained cells (preferred) or CompBeads (dim, rare markers) for compensation calculation for each marker included. In addition, FMO (Fluorescence Minus One) or Isotype Controls are needed for a first panel validation or may be an indispensable control in case of Cytokine Release Assays or inconclusive marker discrimination; this will be discussed after panel validation.
Questions regarding experimental design and controls should be discussed in advance as faulty experimental design is no excuse and will be charged according to the booked time.
Cell size: should not be greater than 1/4th of the nozzle size, larger cells need larger nozzle sizes. The bigger the cell size; the lower expected purities can be achieved due to increased stream fanning. Nozzle changes are time consuming, therefore, users have to book the instruments 2 hours in advance before and after the sort (4 in total), which will be billed accordingly.
Cell Counts and Sample Volume: We recommend a cell density not higher than 5*107 cells per ml for samples. Experiments using lower cell counts per sample should be re-suspended in 400µl of sorting buffer; thereby reducing dead volume for a reasonable time used up for a sample run. Please bring additional sorting buffer for sample dilution or re-flushing the sample tube.
Frequencies: Analysis and sorting of rare events is only possible if large sample volumes (at an appropriate density) are acquired (plan booking time accordingly). Frequencies below 0.1% are considered rare events and should be pre-enriched for cell sorting for good experimental outcomes. Our facility offers a flow based one parametric pre-enrichment step called Speedenrichment, which is also accessible for our users as an additional option to conventional pre-enrichments.
Sort times and achievable purities correlate directly with target cell frequencies!
Sample material containing only a few thousand cells, even single cells, can be sorted for downstream low detection assays. However, in this case no purity measurements after the sort can be offered.Set up: Sorting requires setting up voltages for detection channels; reading in single colors for compensation, worksheet adjustments using the gating strategy and adjusting sort formats accordingly to user needs. These measures add up in time with the complexity of the experimental draft. Set up times vary from two minutes to half an hour. Washing in between samples or before analyzing sort purities takes roughly five minutes and should be scheduled accordingly. In any case necessary set up times will be fully billed by the facility. Daily Clean, instrument performance and QC are pre-set by the facility and are included in billing prices.
Sample flow: Varies between instruments, sort modes and nozzle size for MoFlo Astrios and FACS Aria III. With the default instrument settings samples on MoFlo Astrios can be sorted twice as fast compared to Aria as shown in the table: Link
Sort rate: The maximum event rate for sorters shown in the table should not be surpassed considerably as they strongly correlate with the drop drive frequency to generate acceptable yields and purities. Depending on the instrument, high speed sorting with a 70µm nozzle creates 80,000–100,000 droplets per second. Sorting with larger nozzle sizes decreases drop drive frequencies to 30,000–60,000 drops per second (85 µm nozzle), or even -45.000 (100 µm). For lower nozzle sizes the default cell concentration of 5*107/ml has to be adjusted to maintain sort efficiencies, therefore we always recommend to bring diluent for sort buffers to your appointment.
Nozzle change: requires approval by the operator; For FACS Aria, an additional 1 hour of booking time will be charged, with one appointment per day. On MoFlo Astrios systems, an additional 4 hours of booking time will be charged, with one appointment per day.Dead cell discrimination: Using viability dyes for dead cell detection improves the quality of the sorted sample and validity of downstream assays. Dead cells are known to bind antibodies non-specifically, exclusion of those false positive events enables better and more reliant discrimination of cell populations.
Cell aggregates: Filtering of sample material is mandatory! The use of FACS buffer containing a final concentration of 2 mM EDTA for your sample material is also necessary to avoid clustering of cells over time. Exceptions are only made for following up 10x experiments or other sensitive PCR downstream procedures.
FACS Buffer recipe: PBS (sterile) containing 0.5% (w/v) BSA, 2mM EDTA adjusted at a pH of 7.45
Cells known to having a tendency to form aggregates may be treated in advance with some modifications:
- Use calcium/magnesium free buffer
- add higher concentration of EDTA (>2 - 5 mM)
- use 25 µg/ml DNAse I + 5 mM MgCl2 (without EDTA), if the cell preparation / homogenization induced needs improvement
- consider adding 1% Accutase in buffers
Following the guidelines of the DFG, we are archiving sort fcs files for 10 years. In case worksheet layout as pdf or screenshot and sort report should be archived in addition we ask for special announcements by the users.
Your data can be accessible internaly via the MWN (Münchner Wissenschaftsnetz) webdisc access. External users can ask for data transfer via cloud systems, share and connect or Dropbox. No USB-keys or external harddrives are allowed on the computers!
Final note:
- Cell sorting is provided as a service, instruments for sorting are exclusively operated by the Flow Cytometry Facility staff. Analyzer can be used after introduction and training by us, but still according to our “Concept and Terms of use”.
- We have user rules, please read and abide by them.
- All formal presentations or publications containing data from work performed at our Flow Cytometry Core Facility must be identified. For further information, see Rules for Acknowledgment and Collaboration