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Cell cycle Analysis by Flow Cytometry

Shifts in the redistribution of phases of the cell cycle in response to various stimuli including a response to growth factors, drugs, mutations or nutrients is readily assessed by flow cytometry. Protocols for determining the proportion of cells that are in the G0/G1, S and G2/M phase usually fall into one of three classes, DNA content analysis, DNA analog incorporation experiments, and the use of intracellular markers of the cell cycle. The use of intercalating dyes to measure DNA content are the most common and are usually sufficient for most investigators to determine relative shifts in the phases of the cell cycle.

One of the first problems often associated with the cell cycle is understanding how standard culture techniques effect changes in the cell cycle. Often these changes are not observed until after the first sets of experiments are done. Cell lines can be unknowingly arrested during culturing techniques. Plating cells too densely or having clusters of cells is a common problem that effects the distribution of the cell cycle. Most cell lines are still sensitive to contact inhibition, and a 75% confluent plate is a very subjective observation that can have a major influence on the day to day distribution of the cell cycle. It is also commonly observed that as suspension cells become more dense their growth slows down, this is usually the result of a G1 arrest or an increase in the number of cells in the G1 phase. It should also be noted that when cells are stimulated with a "growth inducer" the relative distribution of the cell cycle may not change even though there is a noted decrease in doubling time.

DNA content experiments only determine the relative distrubution of the cell cycle phases and do not give a mitotic index. Although most cell lines are growing, the amount of time spent in each of cell cycle phases is variable. Most cells spend 50-80% of the their growth time in the G1 phase and the least amount of time in G2/M(less than 10% in some cell lines). It is common to have a cell line with a doubling time of 20hours, with 14hrs spent in G1 and a G2/M phase that only takes 4hours. If the cells are not synchronized in any way and are randomly distributed within the cell cycle-60% of the cells will be G1, 20% will be S phase and 20% will be G2/M.

Cell Cycle Protocols
DNA Content (Intercalating Dyes)	Absolute S phase (DNA Incorporation)	Cell cycle dependent Expression (Intracellular Antigens)
		Reference
Cheap, easy and fast	Correct S phase (Mitotic index)	Allows multiparameter analysis
Requires time courses and apoptosis can be misleading	Often difficult to optimize for your cell type	Requires optimization

DNA content Analysis on the FACSAria (for FACSAria users)
Settings for Optimal Resolution
Sheath pressure set at low(20psi) Reduces cell velocity through interrogation spot	Flow Rate set at 1; keeps inner stream as narrow as possible	70micron Nozzle; keeps inner stream as narrow as possible	Threshold trigger on PI signal; helps with slit scanning (see below).
Samples should be optimized for cell quality and to prevent cell aggregation.			!unable to detect apoptosis with PI trigger!
Filters:	PMT D-575/26 BP Works	PMT D-585/42 BP Better	PMT C-610/20 BP Best
	FCS-2.0 file (Hypotonic PI) (Treated HL60 cells)	FCS-3.0 file (Hypotonic PI) (Treated HL60 cells)	FCS-2.0 file (Hypotonic PI) (MCF7 cells)
FACSAria generated FCS file downloads	These are .FSC files- You may need to change the extension to .fcs when downloading the files.
Unable to download	Try Internet explorer then right click on the "FCS file" link and "save target as" and make sure the extension is .fcs
Area Scaling, Window Extension and slit scanning	When the FACSAria becomes a slit scanner, it is not clear that the DNA content analysis can be done correctly. The small beam geometry of the FACSAria(only 9x81microns) is limiting. Instruments like the calibur and the LSRII have beam spot sizes 3times larger than the FACSAria. This means that samples run the FACSAria exceeding 9microns or the size of a large lymphocyte will be slit scanned. As with all DNA analysis runs, slit scanned particles can only be measured using the pulse area parameter. When samples are slit scanned on the FACSAria some of the DNA content signal is lost, because the top portion of the area curve is flat and some signal is lost and excluded from the area parameter calculation. This lost signal may reduce the G2/M peak because the cells in the G2/M phase are often the largest cells of the population. Since nuclei are smaller than cells, it may help to trigger off from the DNA signal or nuclei to help reduce slit scanning.
Setting area scaling and window extensions	When moving to low pressure your area scaling factor will be very different, most often in the .45-.55 range. The area scaling factor must be set correctly to acquire both the area and width parameters. I have found it useful to use both an area vs height dot plot and to compare area and height histograms for the DNA content signal. NOTE If the area vs height dot plot shows a curve then you have signal that is being slit scanned- a loss of interpreted G2/M phase is likely. I also suspect that if you need to use a window extension greater than "2", you will also be slit scanning and will need to run the samples on a different instrument; although I have not confirmed this.
Additional information The digital software has several advantages including the ability to apply compensation after data collection and is well described by Ben Verwer in the following White paper PDF file. Digital Analysis-FACSDiVa Option - The small beam geometry of the FACSAria(only 9x81microns) is limiting. Instruments like the calibur and the LSRII have beam spot sizes 3times larger the FACSAria. This means that samples run the FACSAria exceeding 9microns or the size of a large lymphocyte will be slit scanned. Slit scanned particles can only be measured using the pulse area parameter. This causes several problems when looking at data collected on the FACSAria including; -loss of resolution -difficult doublet discrimination -difficulty comparing cells of different sizes within the same sample.

Cell Cycle Analysis by DNA Content

DNA Content Protocols
Hypotonic Lysis	Fixation	Viable stains
Most consistent across cell lines	Allows multicolor and apoptosis analysis	Allows cells to be sorted
No Apoptosis	Fixation induces aggregation, may denature GFP, or block ab epitope	Intact chromatin gives reduced resolution
Hypotonic Lysis Buffer (PI) Propidium Iodide 0.025g PI 0.5g Sodium Citrate 0.5ml Triton X-100 to 500ml of dH2O	PI Staining Solution 40ug/ml PI and 100ug/ml RNaseA in PBS	Hoechst stock Soln. 0.112 g Hoechst 33342* 200 ml of dH20 2 ml 95% ethanol Mix thoroughly.

Hypotonic Lysis for DNA content This protocol is usually the easiest and most consistent protocol for many cell types, and can be modified to use any of the DNA intercalating dyes The hypotonic solution lyses the plasma membrane and prevents many of the aggregate problems associated with fixed cells. Propidium Iodide(PI) and 4,6-diamidino-2-phenylindole(DAPI) are frequently used. Simply resuspend 1million washed cells in 0.5ml of Hypotonic staining Solution or add the hypotonic lysis buffer directly to a washed plate. Keep at 4*C in the dark for at least 30min. Remove aggregates by filtration through 40-70um mesh. Samples are then ready to run.

Ethanol Fixation Many cells form aggregates when fixed with ETOH and this is often a difficulty with this protocol, be sure to resuspend cells to a single cell suspension at each step. In addition, some samples will have low sample concentrations after removing aggregates and may have longer run times(5-10min). Aggregated samples can sometimes be improved with a 16gauge needle. It is also useful to provide an extra stained sample for instrument setup, when sample concentrations are low.

1.) Wash a single cell suspension of 1-5million cells in ice cold PBS.

2.) Re-suspend cells in 200ul of PBS/0.1%FBS by vortexing or small tip pipette.

3.) One drop at a time add 4mls of ice cold 70%ETOH to the cells.

4.) Incubate at least 1hr to overnight at 4*C.

5.) Pellet cells and resuspend cells in 1ml of PI solution(40ug/ml PI and 100ug/ml RNaseA).

6.) Incubate cells at 37*C for 1hr.

7.) Filter cells through 40-70um mesh prior to analysis.

Staining viable cells for DNA content. DAPI and the Hoechst dyes have been successfully used to determine cell cycle profiles in live cells. DAPI is more commonly used and both dyes are considered membrane permeable. Of course this is completely dependent on your cell type. Concentration of dye(.5-10ug/ml) and time of incubation(~30-90min) will usually have to be optimized for your cell line. Some cells may require the addition of low concentrations of non ionic detergent. The following are two protocols that will provide a starting point for your cell type.

Viable Hoescht 33342 Stain

1. Cells should be maintained in the media that is normal for their growth.

2. Add 10 ul of 1 mM Hoechst 33342 per ml of media. Incubate for 30 minutes at 37°C.

3. Cells must be maintained in Hoechst, DO NOT wash out the stain.

4. Run samples on the flow cytometer as soon as possible.

* CALBIOCHEM cat.# 382065-Q Bisbenzimide H 33342 Fluorochrome, Trihydrochloride.

1X DAPI (Working Solution) To 500 ml dd H2O add: 8.5g NaCl (final conc. = 146 mM) 1.2 g Tris Base (final conc. = 10 mM) Adjust pH to 7.4 with HCl. Add: 4 ml of 500 mM CaCl2 solution (final conc. = 2 mM) 44 ml of 500 mM MgCl2 solution (final conc. = 22 mM) 50 mg (0.05g) BSA 1 ml nonident P-40 detergent (final conc. = 0.1%) 10 mg DAPI (4,6-diamidino-2-phenylindole*) powder (final conc. = 10 ug/ml) 100 ml DMSO (final conc. = 10%) Add dd H20 to final volume of 1 L. Store in dark or foil wrapped bottle at 2-6o C.

DAPI (4,6-diamidino-2-phenylindole) can also be used.

1. In some cases the cells will be spun down and the supernatant removed before adding DAPI.

2. Resuspend the pellet in at least 200 ul of DAPI. In general you would want the cells at a concentration of 2 x 106/ ml of DAPI. The cells may be able to be run on the flow cytometer immediately.

3. The cells can also be frozen for analysis at a later date.

Cells in suspension are spun and resuspended in a solution of 10mg/ml 4,6-diamidino-2-phenylindole (DAPI) and 0.1% nonidet P-40 detergent in a Tris buffered saline.

*Accurate Chem.Co. #18860

Intercalating Dyes
Dye	Excitation max	Emission Max	Excitation Laser	Comments
Propidium iodide(PI)	536	617	488	best CVs
7-AAD	546	647	488	Compatible with FITC & PE
Ethidium bromide	493	620	488	PI is better
Hoechst 33342/33258	343	480	407(UV)	Membrane Permeable
DAPI	345	455	407(UV)	Membrane Permeable
Pyronin Y(PY)	503	575	488	Binds RNA G0-G1 discrimination compatible w/7-AAD & DAPI

Analyzing Cell cycle FCM data.

Most cell cycle analyses done by Flow Cytometry(FCM) are dependent on intercalating dyes. Shown below are three different analyses types performed on the same sample and FCS file. One of the most important factors in interpreting FCM DNA content data is the quality of staining. This often expressed as a measure of correlation variation of the G1 peak. Relatively good staining will have a CV of less than 5-8% depending how it is measured.

Poor quality histograms can be the result of:
-Cell aggregates -Poor cell quality
-Nonsaturating amounts of PI
-Insufficient fixation
-RNA contamination
-Apoptosis

DNA Content Analysis Types
Histogram	ModFit LT (Verity)	BRDU

Differential analysis of the same FCS file
Histogram	ModFit LT (Verity)	BRDU
G1=59.3%	G1=54.8%	G1=61.3%
S=24.5%	S=34.7%	S=28.3%
G2/M=14.4%	G2/M=10.6%	G2/M=10.6%
Simple and sufficient for most analyses	Very useful with aneuploid populations	Great
-Most subjective and tends to underestimate S phase -Appropriate for comparing relative changes between samples	-Requires "standard" DNA profiles -Difficult to model samples correctly with apoptosis	When you optimize it!