Flow Cytometry Facility

Flow Cytometry is a technique that is used to measure properties of individual cells in a fluid medium. In the Flow Cytometer, cells in solution are passed individually through a measuring point where the sample is illuminated by a light source (most often laser). This allows for a very quick measurement of parameters of a heterogenous mixture of large number of cells (thousands of cells/per second) and also can be used to sort selected cell populations for further study.

The Flow Cytometry lab in the Biology Department has one Flow Cytometry Analyzer (CytoFLEX) and one Fluorescence-Activated Cell Sorter (BD FACSAria2). Students can be trained by the lab technician to use the instruments on their own.

Students at CMU that are performing research under the guidance of faculty advisors have used or currently use the flow cytometers for a variety of projects, including sorting fluorescently labeled neurons, counting algae in lake water, studying cells of the immune system, exploring cell communications in slime molds, and using bacterial models to determine mechanisms of disease.

​The Flow Cytometry Facility is located in the Bioscience building. The facility has two instruments: Beckman Coulter CytoFLEX and BD FACS Aria2. The instruments can be used by trained and authorized users. The facility also has a part-time technician who can run samples for users and train users to use the instruments on their own.

Equipment

Beckman Coulter CytoFLEX

The Beckman Coulter CytoFLEX can be used for flow cytometric analysis. The instrument has two excitation lasers – Blue (488nm), configured to detect two fluorescent parameters (FITC and PE) and Red (638nm), configured to detect two fluorescent parameters (APC and APC-A750). The instrument is controlled by CytExpert Software. In addition to the Forward and Side Scatter detectors, the instrument has the following bandpass filters:

Blue laser

• FITC (525/40)
• PE (585/42)

Red laser

• APC (660/20)
• APC-A750 (780/60)

BD FACS Aria 2

The BD FACS Aria2 can be used for both flow cytometric analysis and cell sorting. The instrument has 3 excitation lasers (Blue - 488nm, Red – 637nm, and Yellow-Green – 561nm).In addition to Forward and Side Scatter detectors, the instrument has the following dichroic mirrors and bandpass filters:

Blue laser

• FITC (505LP; 530/30)
• PerCP Cy5.5 (690LP; 695/40) 
  

Red laser

• APC (670/30)
• APC CY7 (750LP; 780/60) 
  

Yellow-Green laser

• PE (586/15)
• PE TxRd (595LP; 610/20)
• PE Cy5 (635LP; 670/30)
• PE Cy7 (750LP; 780/60) 

Rates

The facility charges $35 per hour for the use of both instruments. The rate is the same whether the technician runs the samples, or the trained user runs the samples. There is also a fixed Set up fee for every time the instrument is used - $15 for the CytoFLEX and $40 for the BD FACS Aria. The billing is done in the beginning of each month for the use of the instruments from the previous month.

CytoFLEX

• Set-up fee: $15
• Rate: $35/hour

BD FACS Aria

• Set-up fee: $40
• Rate: $35/hour

Protocols

Sample Concentration Single Cell Suspension

For sorting of live cells, we suggest a cell density of 2x10⁶ cells/ml – that is the final concentration of cells in your sample to be sorted.

Single Cell Suspensions are required for Flow Cytometry analysis/sorting. The appropriate cell concentration for sorting will vary depending on the sample (cell size, type, etc.). The percent of cells expressing the fluorophore of interest will be a determining factor as to how many ml of cell suspension is needed for the sorting process. If relatively small number of cells are expressing the fluorophore of interest then a bigger starting volume for the sorting will be needed as well as a longer sorting time. Another important factor is how many cells you want to collect from the sort.

The BD FACS Aria 2 can operate with the following nozzle sizes:

Generally, when sorting, the nozzle should be about 5 times the size of the sorted cells – the bigger the cell, the bigger the nozzle.

The cell sorter sorts droplets (which contain the cells of interest). The droplets are generated by vibrations of the stream. The frequency of vibration depends on the nozzle size – the smaller the nozzle size, the higher the frequency; the bigger the nozzle size, the lower the frequency. Ideally, we would like to have an event rate of approximately 1 cell per 4 droplets – this gives the highest probability that there is no cell in the leading or lagging droplet. If the sample is too concentrated the abort rate increases (when a second cell enters the analysis window before the first cell is finished with the processing – the data for both cells is lost. If there are too many events per second there is increased probability of 2 cells in one droplet or cells in the leading and lagging droplets – that compromises the ability to sort and affects the purity and recovery.

Cell Preparation, Media and Sample Filtering

Harvest cells and resuspend in flow cytometer friendly media with 2X antibiotics at approximately 1-2x10⁶ cells/ml final concentration.

• Flow cytometer friendly media – needs to be a clear buffer with low protein content (1%-2%)- listed below is the recipe for basic sorting buffer.
• Supplement the buffer with 2X antibiotics (i.e. pen/strep) – this will help with prevention of contamination during the sorting process
• Samples need to be in 12x75mm, round bottom, 5-ml sterile tubes – tubes are available in the flow cytometry facility, see Megi for access. Tubes can be polypropylene or polystyrene. Also, I suggest keeping the cells on ice when transporting and sorting.
• Samples must be filtered prior to Analyzing or Sorting with a 30-40m filter! This prevents clogging the nozzle of the instrument. When the instrument detects a clog it automatically stops the sorting. Additional time is needed to de-clog the instrument and bring it to sorting status.

It is recommended to bring extra sorting buffer in case a dilution of the sample is needed.

Basic Sorting Buffer Recipe

• 1x PBS (Phosphate Buffered Saline - Ca/Mg++ free)
• 1mM EDTA
• 25mM HEPES pH 7.0
• 1% Fetal Bovine Serum (Heat-Inactivated, Ca/Mg++ free)
• Sterilize by passing through a 0.2µmfilter and store at 4^oC.

Notes: The use of Ca/Mg++ free buffer helps with reducing the cell aggregation. The addition of protein source is minimal – it should be just enough to keep the cells happy; 0.1%-1% BSA can be used instead of FBS. BSA helps with decreasing the autofluorescence. Addition of chelators like EDTA helps reduce cell adhesion (this prevents clumping of cells and reduces the chances for clogging the instrument). The addition of HEPES helps with maintaining pH stability (the high-pressure during sorting compromises buffer capacity).

Buffer Modification for Different Cells

1. For Clean Lymphoid Cells: The buffer can be simplified to just HBSS with 1% FBS. The additional cations in the recipe promote better viability. Since these cells are not prone to clumping, the lack of EDTA is not a problem.
2. For Sticky Cells: Raise the concentration of the EDTA to 5mM and use FBS that has been dialyzed against Ca/Mg++ free PBS. Chelators (EDTA) help reduce cation dependent cell to cell interaction and adhesion.
3. For Adherent Cells: Typically, trypsin (or other detachment buffer) is quenched with culture media or a PBS/FBS buffer. This reintroduces the cations that facilitate the cells reattaching to the plate (or each other). Use Ca/Mg++-free FBS buffer in order to stop the detachment. Additionally, the level of EDTA can be increased if necessary – up to 5mM (but too much EDTA can kill the cells).
4. For Samples with High Percentage of Dead Cells: If there are a large number of dead cells in your sample, it is likely that there is soluble DNA from the dead cells that will come out of solution. This DNA will start to coat the cells and lead to severe clumping. The addition of 10U/ml DNA-ase to the buffer recipe will help reduce DNA associated clumps.

Pre-sort Analysis

We need to perform a pre-sort analysis to determine the percent of cells expressing the fluorophore of interest. Expressing cells will be gated for the population of interest to be sorted. Unstained or non-expressing cells need to be used as a negative control.

For the pre-sort analysis you will need 2 tubes:

• 1. unstained / non-expressing cells ~ 300 µm 
• 2. cells with expression of interest ~ 300 µm

Collecting Media and Collecting the Sorted Cells

C for at least 1 hour prior to sorting. Pour the serum out (it can be reused for up to a month) and add the media. RNA/DNA lysis buffer can be used as a collection media when needed.^oThe sorting process is harsh on cells and recovery seems to be quickest when cells are sorted into tubes containing the normal growth media. Cells are sorted in droplets of sheath fluid that mix with the media in the collecting tubes. The media in the collecting tubes should be a PBS or HEPES buffered media with high protein content (20% FBS or BSA, up to 50%) and double amount (usually 2%) of antibiotic (pen/strep or gentamicin). It is a good idea to coat the tubes with BSA or FBS prior to sorting into them, especially the conical 15ml centrifuge tubes. This will prevent the sorted cells from sticking to the tube walls and drying out. To coat the tubes, fill them with 4% BSA (in 1xPBS – filter sterilized) or pure FBS and incubate them at 4
For the sorting you will need:

• Cells in flow friendly media sample #1 ~ 1.5ml @ 2x10⁶ cells/ml final concentration in cytometry tube.
• Cells in flow friendly media sample #2 ~ 1.5ml @ 2x106 cells/ml final concentration in cytometry tube.
• Collection tubes with growth media with 2x antibiotics.

When sorting with the 70 µm nozzle, 1ml of sorted volume will have ~ 1,000,000 cells.

When sorting with the 100µm nozzle, 3ml of sorted volume will have ~ 1,000,000 cells.

1-2 different populations simultaneously can be sorted into:

• 15ml conical tubes (with 3ml collection media).
• 12x75mm round bottom tubes (with 1ml collecting media).

3-4 different populations simultaneously can be sorted into:

• 12x75mm round bottom tubes (with 1ml collecting media).
• 1.5ml Eppendorf tubes (with 500 l collecting media).

The type of collection tubes is chosen based on the number of cells you expect to collect and the nozzle size

* Polypropylene tubes are recommended as collecting tubes for the BD FACS Aria. The instrument charges the stream before sorting the cells and a charge can build around the polystyrene tubes.

It is recommended to bring extra sorting media and extra tubes.

Post-sort Analysis

Representative samples from post-sorted cells can be ran to determine the effectiveness of the cell sort.

Controls

In addition to your experiment controls, please consider the following controls, needed for setting proper voltages, compensation and gating of the population of interest.

Unstained Cells Unstained (or un-expressing) cells are needed to determine where the negative population is. They help to determine the level of background fluorescence/autofluorescence and allow to set proper voltages and negative gates.

Viability Controls With sorting it is strongly recommended that dead cells exclusion dye is used to distinguish between dead/live cells and to make sure that only viable cells are analyzed and collected. This can be a DNA-binding dye (like PI or 7-AAD) or variety of protein-binding dyes (they are fixable). Dead cells have greater autofluorescence and increased non-specific antibody binding – thus they can greatly affect the staining and respectively the quality of the data. The presence of dead cells in the sample can lead to false positives and can reduce the dynamic range.

Single Stain and Compensation Controls When performing a multicolor flow cytometry experiments it is necessary to perform a compensation for fluorophores with overlapping emission spectra. In this case a single stain control is needed for each fluorophore used. Antibody capture beads can be used instead of cells. There are several factors that have to be considered:

• Each single stain control has to be brighter or at least as bright as the sample.
• The autofluorescence of both positive and negative control populations should be the same.
• The same fluorophore must be used both for the compensation control and for the sample.

Fluorescence Minus One (FMO) Controls In multicolor flow cytometry panels FMO controls help to determine the proper setting of the gates. This is important when identifying a positive from negative population and when the expression levels are low. FMO controls contain the sample and all the fluorophores used minus one.