Sorting FAQs

What sorter should I book?

--> That, of course, strongly depends on your experiment. See the sorter’s configurations for further details. So for detection of Dapi, Hoechst or Brilliant UV (BUV)- and BrilliantViolet(BV) dyes, Pacific blue, Pacific Orange, V500, V450..., you can only use the Fusion. Propidium iodide (PI), CFP, YFP, Venus, GFP, mCherry, dTomato, RFP, dsRed, PE, FITC, PerCP, APC, Alexa647, Alexa633, Alexa488, APC-Cy7, Pe-Cy7 among others can be detected on both FACS machines.

What do I need to bring with me to my sort appointment?

--> Samples: pre-filtered through an at least 70 µm cell starainer (sorting samples and your controls)

--> Vessel(s) to collect into with fluid (media/PBS)

Which controls do I need?

--> We recommend the use of unlabeled cells, single stained tubes and FMO controls in addition to experimental controls for most (immuno)phenotyping projects

--> DNA cell cycle projects usually do not require unlabeled cells

--> Questions regarding experimental design should be discussed with us prior to experimentation

What can I sort into?

--> You can collect your cells/particles into 2 mL or 1.5 mL Eppendorf tubes, 5 mL FACS tubes (4 populations simultaneously) OR 15 mL Falcon tubes (2 populations simultaneously) OR into one plate at a time (6/12/24/96/384)

How many cells should I bring?

--> The number of cells to prepare for flow cytometry experiments can vary drastically and depends heavily upon the detection assay and purpose of flow cytometric experiment

--> Beginning with a surplus of cells is ideal; however, source size may be the limiting factor. For cell analysis, beginning with 1 x 10^6 cells is always a good place to start but less is also ok (sample volume should exceed 100 µL but please don’t dilute cells more than necessary.

--> Rare event analysis can only be achieved if large samples are taken and measured (plan booking time accordingly)

What density should the cells be at?

--> Cells should be at approx. 10-20 million/mL. Please bring extra buffer to dilute out the samples if it turns out it is too concentrated. Note, as a rule of thumb, 1 mL sample volume will take approx. 1 hour sorting time (with our standard 85 µm Nozzle).

How many populations can I collect at once?

--> Our sorters can sort into max. two 15 mL OR max. four micro tubes OR four 5 mL facs tubes at one time. They are designed to collect only one population/ one well at a time for any of the plate configurations.

How much time should I reserve?

--> We need sufficient time for setup, sample analysis/sort, and for cleanup. SETUP: 15 min plus time to run compensation controls and setup template (10-15 min, can be longer in case of many comp. controls or low bead/cell numbers). SAMPLE: Rule of thumb: 1 mL sample will require 50 min run time/sorting time (lowest flow rate, 85 µm Nozzle). CLEANUP: 10 min.

Why should I stain dead cells?

--> Staining your sample for dead cells will improve the quality of sorted sample. If dead cells are labeled and removed from the sorted population, the user will end up with more viable cells to perform downstream experimentation. Besides, dead cells tend to bind antibodies unspecifically, consequently you will be analyzing/sorting false positives.

Which sheath buffer is used in the cell sorters?

--> We use the Beckman Coulter Isoton II diluent. The ISOTON II diluent is a filtered, phosphate-buffered saline solution compatible with most human and rodent cell types, and may be used for the suspension of most biological cells.

What is the maximum sort rate?

--> The maximum event rate for purification on the sorters should not surpass ¼ of the drop drive frequency. High speed sorting creates 60,000–90,000 droplets per second. Low speed sorting creates 30,000–50,000 drops per second. Special setups using atypical nozzles (100 µm, 130 µm) will deviate from these settings. In all cases, the cell concentration is adjusted to maintain sort efficiencies of 75-80% or usually better (>90%). We recommend diluent is brought to the appointment.

Is the sort sterile?

--> The sheath in our sorters is filtered through a 0.2 µm filter and the sample lines are regularly cleaned with BD FACS Clean, detergent and water, while surfaces are regularly liberally wiped with 70% ethanol. However, our sorters are open to the air and are classed as clean and not sterile. Therefore if you want to culture your cells after sorting, we advise you to add pen/strep to your media.

How do I access my data?

--> You can download your files under: \\project\FlowCytometryLab\username. USB sticks or any other media are strictly forbidden on the machine’s PCs.

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