Flow Cytometry

Flow Cytometry Resource Room 2221:

The CORE provides training and access to analytical flow cytometers, BD FACSCalibur and BD LSRFortessa with HTS plate reader. The CORE also processes biological samples and FlowJo data in addition to providing cell sorting services on the BD Aria II. The CORE offers technical support during experimental design, optimization, and data analysis as well.


Assisted-use hours occur from 8:30 am to 5:30 pm. Users must schedule cytometers or services in advance with Todd or Josh (at least three days in advance for cell sorting). Unassisted self-use of the cytometers is possible 24/7 for trained users with prior approval.

What is flow cytometry?

Flow cytometry is a laser-based analytical tool that measures the size, internal complexity, and fluorescence of cells in suspension as they pass a laser light source at high speed. Fluorescently labels probes or dyes can be added to the sample to determine specific physical and/or biochemical properties. The cell-sorting function of flow cytometry physically separates these cells into different subpopulations based on the presence and intensity of fluorescence markers.


  • BD Aria II: Five laser, fifteen color cell sorter. Sorts into tubes, well plates, or onto microscope slides
  • BD FACSCalibur: Two laser, four color analyzer.
  • BD LSRFortessa: Five laser, sixteen color cell analyzer.

Common uses:

  • Cell cycle and apoptosis/cell death assays
  • Detection of one or more fluorescent proteins, including GFP and many others
  • Cell phenotyping with fluorescently tagged monoclonal antibodies
  • Cell sorting (segregates a heterogeneous mix of cells into distinct subtypes — to re-culture or analyze them)


Frequently Asked Questions for Cell Sorting:

1. What information should be provided to the CORE before a sort?

  • Cell type (species, tissue, primary or cell line)
  • Approximate cell size
  • Which fluorophores have been used to label the cells
  • Number of populations to be sorted (1-4)

2. What should you bring to a cell sorting appointment?

  • Samples pre-filtered through a 40-micron mesh
  • Pre-labelled vessel(s) with buffered media inside for collection

3. What type of cells can be sorted?

Any cells in single cell suspension can theoretically be sorted. Cells that aggregate or settle out of suspension can be difficult to sort. Primary human samples or samples infected with BSL2 pathogens require special handling and prior approval by the CORE before sorting.


4. What collection vessels can be used?

You can collect into 5-mL 12×75 tubes, 15-mL Falcon tubes, or into well-plates (6/12/24/96-well). You can also sort onto slides.


5. How many cells are needed?

This number will depend on 1) how many cells you need to acquire, 2) the frequency of the target population, and 3) the yield (typically 75-90%). For example, if the target cells comprise 10% of your unsorted population and you need to recover 106 target cells, you would need approximately 2 x 107 cells in your sort sample.

106/(0.1 x 0.5) = 2 x 107 … Using a 50% yield rate is a worst-case scenario that ensures enough target cells are collected.


6. What cell density should the sample be?

Cells should be at approximately 10 million/mL. Bring extra buffer to dilute the sample in case it’s too concentrated.


7. Why is the yield less than 100%?

Yield is reduced by …

  • Loss of target cells due to cell death or adherence to tube walls
  • Sort conflicts: target and non-target cells occur simultaneously, so both cells are rejected for sorting to avoid compromising the collected-sample purity
  • Electronic aborts: cells arrive in the laser beam too close to one another to be sorted

8. How can cell clumping be avoided?

Keeping cells in single-cell suspension is critical to the success of a sort. Cell preparations with a large number of aggregates can cause the nozzle to clog, which requires additional cleaning or adjustment. A clog also increases the possibility of sort contaminations. Cell clumping can be an issue with adherent cells, activated cells, or with samples containing a high percentage of dead cells. Raising the EDTA concentration to 5 mM in the sample buffer may help to reduce cation-dependent cell-to-cell adhesion. For adherent cells, commercially-available cell detachment products like Accustase can reduce clumping. Regardless, the sample should always be passed through a filter to remove as many aggregates and debris as possible before the sort’s start.