University of Vermont

College of Medicine

Vermont Cancer Center

VCC Flow Cytometry Facility

Flow Cytometry Facility

Flow cytometry allows researchers to analyze cells or cellular components for a wide range of immunologic, intracellular, genetic, and morphological characteristics. The flow cytometry instrument uses a flowing stream of isotonic sheath fluid to carry suspended cells through a flow cell and into the path of a laser beam. Once in the beam, the cell will generate light signals that are detected by six detectors. Two of the detectors, forward scatter and side scatter, provide information on the size and granularity of the cell respectively. The remaining four detectors are wavelength specific and collect signals emitted by cells that have been tagged with fluorescent dye.

Flow cytometry is extremely powerful and can lend itself to a wide range of applications including, but not limited to: DNA/RNA analysis, cell cycle analysis, intracellular antigen measurement, DNA ploidy analysis, cytokine detection, apoptosis, reticulocyte analysis, immunophenotyping, platelet analysis, phagocytosis, cell proliferaion assays, intracellular pH, cell enumeration and sizing, intracellular calcium, cellular viability, and oxidative burst. This is only a partial list and many other assays can be done. The facility actively participates in new protocol development, and encourages investigators to inquire about additional assays or applications. Please call if you have questions.

Instrumentation & Software

  • EPICS XL / XL-MCL Flow Cytometry System with 488 nm Argon laser
  • FlowCentre™ Multimedia Workstation with SYSTEM II and EXPO320™ Software
  • Dell XPS workstation for post data analysis
  • WinList 4.0 Verity software house
  • ModFit versions 2.0 and 3.0 Verity software house
  • WinMDI 2.8 Scripps Research Institute

Cell Cycle Analysis Using Propidium Iodide

Propidium Iodide stains double-stranded nucleic acids stoichiometrically. It stains both RNA and DNA, so we need to treat the cells with RNAse A in order to stain only the DNA. PI absorbs 488 nm light, and has a peak emission at 617 nm with a broad range from 550 to 720 nm. PI fluorescence is detected with the Epics XL’s “FL3” PMT which senses light at 620nm. It can also be sensed on the FL1, FL2 and FL4 detectors, so if you are staining with another fluorochrome, color compensation is required.

Reagents

  • PBS
  • PBS with 1% FCS
  • Stock 50X PI solution: 0.5 mg / ml in 38mM sodium citrate, pH 7.00, stored at RT in foil wrapped tube. (Sigma #P-4170) WARNING: TOXIC and light sensitive.
  • Stock 40X RNAse A: 10 mg / ml, Pharmacia #27-0323, 100 mg, Ribonuclease I “A”, Bovine pancreas, 82.5 U / mg
    Dissolve at 10 mg / ml in sterile ddH20.
    Aliquot into microfuge tubes, 500 µl each.
    Boil 10-30 minutes to kill DNAse.
    Store at -20°C.
  • PI Working Solution: 80 ul of RNAse Solution, 100 ul of PI solution, 19.8 ml of PBS with 1% FCS

Harvesting Adherent Cells

  1. Warm media (complete media with FCS), trypsin-EDTA, and PBS in 37°C water bath.
  2. In the tissue culture hood, aspirate the media out of the plates with a clean pasteur pipet and the vacuum flask setup.
  3. Pour about 10 ml PBS into the plate (for a 10 cm plate), aspirate PBS.
  4. Repeat PBS wash.
  5. Add 1 ml trypsin-EDTA to each plate.
  6. Incubate ~ 3 min in the 37°C incubator.
  7. Spank plates. Add 9 ml of media to inactivate the trypsin.
  8. Rinse the plate several times with the media / cell suspension to loosen all of the cells, then transfer the cells to a 15 ml conical tube.
  9. To obtain more cells, rinse the plate with ~ 3 ml of fresh media and add this to the cells in the tubes.

Harvesting Suspended Cells

  1. Pipet the cell suspensions up and down to loosen any cells that have settled to the bottom of the plates.
  2. Transfer cells to 15 ml conical tubes.
  3. To obtain more cells, rinse the plate with ~ 3 ml of PBS and add this to the cells in the tubes.

Staining with PI

  1. Spin cells at 1000-2000 rpm (210-500 G), 3 min, 15°C.
  2. Wash cells 1 time with PBS.
  3. Resuspend cells in 500 µl PBS. (a very important step to avoid clumping!)
  4. Add 7.5 ml -20°C 80% EtOH dropwise while vortexing.
  5. Incubate cells at -20°C.
  6. Spin the cells 1400 rpm (400 G), 5 min, 15°C.
  7. Resuspend cells in 5 –10 ml PBS containing 1% FCS.
  8. Count the cells. Adjust count to 100,000 to 400,000. Also make a mental note of the amount of cell clumping and presence of debris by examining under the microscope. Spin 1400 rpm (400 G), 5 min, 15°C.
  9. Aspirate.
  10. Resuspend the cell pellets in PI / RNAse / 1% FCS / PBS.
  11. The maximum cell density for saturation of the DNA with PI, is < 1 x 106 / ml.
  12. The minimum volume that can be run on the Epics XL is 900 µl. If you are setting up a new protocol, 1500 µl of your positive and negative controls is usually necessary.
  13. Incubate 37°C, 30 minutes and protect the tubes from light.
  14. Store the samples at 4°C until you’re ready to run them on the flow cytometer. Leaving the cells at 4°C overnight improves the cell cycle data significantly. Analyze with in two days.
  15. IMPORTANT: Filter the samples through 53 µm nylon mesh before running on the flow cytometer or you may risk clogging the flow cell.

FACS sorting of cells for RNA isolation

Please consult with the microarray and flow cytometry staff for successful RNA recovery from cells sorted using flow cytometry. Several key points are highlighted below:

Instrument decontamination

Preparing the flow cytometer is often not a small task as it should be completely free of RNases from the sheath tank to the sorting nozzle before sorting. This decontamination procedure may take considerable time depending on what the flow cytometer has been used for, the age of the instrument, and consequently the contamination level. Ensure the dip tube, septa, flow cell, all tubing lines, and nozzles have been decontaminated with bleach, RNase ZAP, ethanol, autoclaving, or other qualifying technique prior to the sort. The sheath fluid and tank must be RNase-free as well. An in-line 0.2uM filter is not sufficient for "filtering out" RNases. Immediately before the sorting run, run a tube of 10% bleach for 300 seconds followed by a minimum of 3 tubes of DEPC water for 180 seconds each.

It should be noted that some operators do not decontaminate their flow cytometer prior to a RNA sort. Instead they run several tubes of bleach and DEPC water and have good results. Again, this is dependent on the contamination level of the instrument. If this approach is used, it MUST be tested on non-critical cells first to determine the final RNA quality. If the RNA is degraded, full instrument decontamination is recommended.

As a rule, retain some cells and extract the RNA to determine the condition of RNA prior to the sort.

During the sort, it is recommended to sort directly into your extraction reagent, such as Trizol LS or Qiagen's RLT (when volumes permit…less then or equal to 500ul). When sorting volumes are high [>0.5ml], it maybe necessary to sort into cold media or PBS and spin your cells to a pellet and freeze or extract using standard procedures. If secondary processing is required, it is important to sort into RNase-free tubes and buffers and keep on ice during the sort. Always extract RNA as soon as possible.

Trizol TM LS

When sorting into Trizol, consider using Trizol LS (cat #10296-010) which is more concentrated than regular Trizol. This formula allows lower quantities of reagent to be used relative to the amount to the sorted sample volume (regular Trizol can tolerate 10% sorted sample or 100ul of total sorted volume to 900 ul of Trizol while TriZol LS allows 30% sample volume). It is rarely applicable to use standard Trizol for most sorting purposes.

Simple Method:

  1. Start with 500ul of Trizol LS in a sterile FACS tube.
  2. Sort into the tube, periodically mixing if necessary to get liquid off sides of tube and keep evenly mixed.
  3. After the sort, using a pipet with sterile tip, measure the final volume.
  4. Calculate the exact volume of sample sorted into the Trizol LS.
  5. Adjust the amount of Trizol LS required to maintain your sample at less than or equal to 30% of the total volume.
  6. Proceed to step 2 of the Trizol protocol.
  7. For the final precipitation step, consider using the Axygen MCT175 Ultra-clear microfuge tubes for best visualization. A co-precipitate maybe added during the final precipitation such as glycogen or Pellet Paint.

It is not recommended to freeze the sample-Trizol mix. It should be extracted ASAP. Only when absolutely necessary, freezing at -80 is tolerable. We have noted as much as a 50% reduction in RNA recovery when extracting previously frozen cells in extraction buffer.

Qiagen's RNeasy

In the case of Guanidium Isothiocyanate (or Qiagen's RLT buffer), sorted at a ratio of 100ul of sort sample to 350 ul of RLT or a multiple of that. It should be noted that when using RLT buffer, that recoveries are often half that of Trizol LS and you lose the transcripts that are smaller than 200 bp, but the RNA is much cleaner. This is especially important with miRNA as they are less than 30 bp and fall into to this <200bp category.

Simple Method using the RNeasy Clean-up protocol

  1. Start with 500ul of RLT Buffer in a sterile FACS tube.
  2. Sort into the tube, periodically mixing if necessary to get liquid off sides of tube and keep evenly mixed.
  3. After the sort, using a pipette with sterile tip, measure the final volume.
  4. Calculate the exact volume of sample sorted into the RLT.
  5. Adjust the amount of RLT required so that there is exactly 350 ul of RLT to every 100 ul of sorted sample volume. (See RNA clean-up protocol in Qiagen handbook)
  6. Add 250 ul of 100% ETOH for every 350 ul RLT-100ul sample volume.
  7. Mix and apply to a RNeasy mini or RNeasy minelute column in 700ul aliquots and spin at > 8000 g. Additional volumes maybe added to the same columns.
  8. If a large amount of preparation is to be added to a column (>5ml), multiple columns maybe used. In this case, the elution of RNA from the columns should be done with the same aliquot of water to maintain a concentrated sample in the final extract. In other word, the 30-50ul of water used to elute your first column will be used to elute your second column and third column. This will maintain your RNA in one 30-50 ul aliquot. This is only necessary for sample containing low concentrations of RNA.
  9. DNase treatment is optional, but expect a 20-30% loss in RNA recovery.

Notes on RNeasy technique:

If the sorted volume is less than 100 ul add DEPC H20 so the amount of sorted sample volume and DEPC is 143 ul. This maintains the 3.5 RLT to 1 aqueous ratio (500/143=3.5). The volume of ETOH must also be adjusted.

The RNeasy Mini kit (74103) is designed to elute the RNA in 30-50ul of final volume and is better suited for higher RNA amounts (i.e. >20,000 cells). For fewer cells, the RNeasy MinElute (74204) is better suited as the final RNA is eluted in 14ul. Both allow an optional DNase treatment step.

It is not recommended to freeze the sample sample-RLT mix. It should be extracted ASAP. Only when absolutely necessary, freezing at -80 is tolerable. We have noted a 80% reduction in RNA recovery when extracting previously frozen cells in RLT extraction buffer.

Comments:

  • We highly recommend to use of Axygen's Ultra-clear MCT-175C tubes and Pellet Paint [EMD Biosciences Cat # 69049-3] for precipitation reactions.
  • When performing on-column DNase treatment, a 30% loss of RNA maybe observed. For maximum recovery from limited sample amounts, on-column DNase treatment should be avoided [if optional]. If this method is employed, ensure that RW1 buffer is added to the column while the DNase is present. Do not spin the DNase through and then add RW1. Omitting this step will result in much lower yields.
  • Using control cells [non-critical cells], conduct a test run through the entire procedure. Do not start with your "good" preps. Quantitate the RNA with the Nanodrop ND1000 and evaluate using the Agilent 2100 bioanalyzer. If quantitation is too low for a Nanodrop reading, run on the bioanalyzer Picochip.
  • Freezing cells in Trizol at –80 is not recommended but is tolerable. Expect a 50% reduction in RNA recovery. This is not the case with Qiagens RLT buffer. We do not recommend freezing RLT with your cells.
  • In rare cases, the flow cytometer, even after a good decontamination procedure can still be a source of RNases. If possible you may wish to consider suspending cells in RNAlater prior to the sort. If RNAlater is not an option, you may consider adding an RNase inhibitor to cell suspension. Of course this is volume dependent and may not be economically realistic.
  • When not sorting directly into extraction reagent, sort into vessels containing media or PBS on ice when applicable. Immediately after the sort, extract RNA according to the selected reagents manufacturer's protocol and evaluate the integrity of the "before" and "after" RNA.
  • Keep nozzle pressure low if possible.
  • When staining cells with any stain including mAb, make sure they are RNase-free and the procedures are RNase-free, this includes all rinse buffers and tubes. If necessary, mix some good RNA with your stain and incubate for 5 minutes and run a RNA assessment.
  • Prior to sorting or during a sort, it may be a good idea to stain the cells with a viability dye so dead cells can be rejected. Dead cells can be a large contributor of RNases.
  • Remember DEPC water DOES NOT inactivate RNases, and is only RNase-free water. Use only sterile RNase-free tubes on the cytometer that have never been open to contaminated air.
  • Routinely we have had success with the RNeasy MinElute cleanup kit [Cat # 74204] by sorting directly into RLT, and extracting immediately using the correct RLT/ethanol ratio, and eluting off the column in 12 ul of DEPC water. However, it is recommended to omit the carrier –tRNA step.

Article of Interest: Biotechniques. 2002 Apr; 32(4):888-90, 892, 894, 896. High-quality RNA and DNA from flow cytometrically sorted human epithelial cells and tissues. Barrett MT, Glogovac J, Prevo LJ, Reid BJ, Porter P, Rabinovitch PS. Fred Hutchinson Cancer Research Center, Seattle, Washington, US

Last modified November 21 2013 02:50 PM