Separation of Cell Populations
Synchronized in Cell Cycle Phase
by Centrifugal Elutriation
Centrifugal elutriation is the only method whereby
large numbers of cells can be separated rapidly on the
basis of size (Diamond, 1991; Merrill, 1998; Davis et al.
2001). The capability of discriminating between very
small differences in cell size provides the ability to separate
cells into sequential cell cycle phase populations
of relatively high purity without the use of drugs
or inhibitors (Bludau et al.
, 1986; Braunstein et al.
1982; Hann et al.
, 1985; Iqbal et al.
, 1984; Wu et al.
1993; Brown and Schildkraut, 1979; Bialkowski and
Kasprzak, 2000; Datta and Long, 2002; Deacon et al.
2002; Hengstschlager et al.
, 1999; Houser et al.
Karas et al.
, 1999; Rehak et al.
, 2000; Sugikawa et al.
1999; Syljuasen and McBride, 1999; Van Leeuwen-Stok et al.
, 1998). It has been used successfully to separate a
wide variety of cell types from suspension and
substrate-dependent cultures and to separate mixed
cell populations liberated directly from tissues or body
fluids (Boerma et al.
, 2002; Dagher et al.
, 2002; Wong et
al., 2001, 2002). The purity of the samples is relatively
high and the cells proceed to grow, following separation,
without a detectable lag period. Thus, centrifugal
elutriation combines speed of separation of large
numbers of cells with little or no perturbation of
the cell growth cycle and avoids the use of agents
that might induce artifact. As additional advantages,
centrifugal elutriation overcomes the limits on cell
number imposed by fluorescence-activated cell sorting
and the long separation times required for unit gravity
sedimentation, as well as problems associated with
osmotic stress in centrifugation media. The only real
compromise is that the purity of the samples is somewhat
lower than commonly achieved with alternative
methods. The developmental history and theory
of centrifugal elutriation are reviewed elsewhere
(Conkie, 1985; Beckman Instruments, 1990).
Centrifugal elutriation was developed by Lindahl
(1948) for the separation of cells and particulate fractions
of cells (Lindahl and Nyberg, 1955; Lindahl, 1986)
based on the original work of Lindbergh (1932). Counterstreaming
centrifugation was used by these investigators
to separate a variety of cell types but was not
generally available to other laboratories until the
introduction of a commercial centrifugal elutriator by
Beckman Instruments in 1973 (reviewed in Beckman
Instruments, 1990). Once available commercially,
centrifugal elutriation was applied to the separation
of many kinds of cells, including bacteria, yeast,
and mammalian cells, grown in culture or liberated
directly from tissues and solid tumors (reviewed in
Beckman Instruments, 1990). In addition, elutriation
has been used to separate cells in different phases of
the cell cycle based on the small differences in size as
cells gradually grow between divisions (Bird et al.
1996a,b). In all cases, successful separation is dependent
on complete dissociation of the cells to a single
cell suspension. Failure to accomplish this affects the
quality of separation as well as cell yield and thus cell
clumps must be removed or dissociated prior to
Centrifugal elutriation imposes two opposing forces
on mixed cell populations to facilitate their fractionation
into subpopulations (Fig. 1 and see Section IV). This technology has proven to be effective in fractionating
cells, based on very small differences in cell size,
with nominal cross contamination, and in numbers
unmatched by other methods of cell separation. In
addition, centrifugal elutriation can be performed
rapidly, requiring only a few minutes (usually 20-
120min) to affect separation and this occurs in media
containing no special additives that might affect the
osmolarity or viscosity of the medium. Thus, with very
little physiological change perceived by the cells, separation
can be affected rapidly. Some shear force is
exerted on the cells during separation, but this is not
sufficient to appreciably affect viability or behavior of
the cells in most cases. Compared to other methods of
separation, such as fluorescence-activated cell sorting
or unit gravity separation, centrifugal elutriation is by
far the most gentle and the most rapid in manipulation
and separation of the cell populations.
|FIGURE 1 Cell separation dynamics and opposing forces in the elutriation chamber. The flow of media
is opposed by centrifugal force in a balance that holds particles in equilibrium in the separation chamber.
Due to different levels of force applied proportional to surface area presented, smallest particles sort to the
inside of the chamber relative to larger particles. Separation is achieved by increasing the flow rate incrementally
to push the smallest population of particles past the widest part of the chamber and into the inner
narrowing section where media flow accelerates affecting elutriation of the population.
In this example application, synchronous fractions
of HeLa S3 cells were analyzed, following centrifugal
elutriation, by flow cytometry and [3
incorporation into acid-precipitable materials (Wu et
al., 1993; Pai and Bird, 1994). Both means of analysis
demonstrated that sequential fractions of elutriated
cells represent sequential cell cycle phases as determined
by the analysis of cell volume, DNA content,
and ability to incorporate thymidine during five
sequential 1-h periods following return to culture.
From this analysis, cells collected at flow rates of 21-
25ml/min were designated the G1
cells collected at flow rates of 29-35 ml/min were designated
the S-phase population, and cells collected at
flow rates of 43ml/min were designated the G2
population. Flow cytometric analysis, based on
measurements of DNA content and cell volume, were
also used to determine the level of contamination of
S-phase cells in the Gl-phase fractions (approximately
3%) and G1
-phase cells in the S-phase cell population
(approximately 10%) (Hann et al.
, 1985). Thus, large
populations of cells were separated rapidly into seven
to eight synchronous fractions without the use of
drugs, with little evidence of perturbation, and with
low levels of contaminating cells.
II. MATERIALS AND
The centrifugal elutriator is from Beckman Instruments
(elutriator rotor assembly Cat. No. JE-6B was
run in a Model J2-21 elutriation centrifuge). Accessories
are used as specified by the manufacturer
throughou t, and the rotor is equipped with a standard
separation chamber. A Masterflex digital peristaltic
pump (0-100rpm Model 07523-70 fitted with a model
7014-21 pump head, Cole-Parmer) is used to pump
cells and media through the rotor.
Materials for cell culture include α-modification of
Eagle's minimal essential growth medium (α-MEM, Invitrogen, Cat. No. 11900-073), fetal bovine serum
(FBS, Hyclone, Cat. No. SH30070.03), donor horse
serum (DHS, ICN Biomedicals, Inc., Cat. No. 29-211-
49), 100× antibiotic/antimycotic solution (Invitrogen,
Cat. No. 15240-062), 10× trypsin solution (Invitrogen,
Cat. No. 15090-046), and Hanks' balanced salt solution
(Sigma, Cat. No. H9394). All plasticware is tissue
culture grade (Corning Plasticware, Fisher Scientific).
All other reagents are standard reagent grade and
available from numerous sources. The water used
throughout this procedure is ultrapure in quality and
is prepared by ion-exchange chromatography
(Barnstead Nanopure) to 18-MΩ resistance and then
glass distilled to remove residual endotoxin and
RNase activity. Solutions are sterilized by autoclaving
or ultrafiltration (0.2 µm).
A. Cell Culture
Growth medium and elutriation medium are used
per standard protocols for the growth of HeLa cells
(Pai and Bird, 1992). If different cell lines are to be
elutriated, appropriate media should be substituted
(see Section V).
- Growth and elutriation media: Dissolve powdered
α-MEM (10-liter pack) in ultrapure water containing a
final concentration of 1× antibiotics and 22.2 g NaHCO3 and make up to a total volume of 10 liters. Place the
medium in a pressurizable vessel (Millipore), filter
through a 0.2-µm filter (Corning) by positive pressure
into sterile 0.5-liter bottles, and store at 4°C. Prior to
use, add FBS (10%, v/v) to make growth medium or
add DHS (5%, v/v) to make elutriation medium. Use
of DHS reduces the cost of elutriation greatly without
detectable effects on cell growth or quality of the fractionation
(see Section V).
- 0.5M HEPES: Dissolve 14.17g HEPES buffer
(Fisher, Cat. No. BP310-100) in ~80 ml water and adjust
pH to 7.2. Adjust volume to 100 ml, filter sterilize, and
store in the cold at 4°C.
- 0.5M Na2EDTA: Begin to dissolve 14.61 g EDTAfree
acid (Fisher, Cat. No. BPl18-500) in ~40ml water
with a magnetic stir bar. Monitor pH of the solution
continuously while slowly adding 1M NaOH (40g/
liter for 1M stock) dropwise. Continue to adjust the
pH up to ~8.0. As the EDTA dissolves, the pH will
continue to fall. Carefully adjust the final pH to 8.0
without exceeding this value. Adjust volume to 100 ml,
filter sterilize, and store at 4°C.
- Trypsin solution: Add 8ml of 2.5% trypsin stock
solution (10 ×), 2 ml 0.5M HEPES, pH 7.2, and 2 ml
0.5M Na2EDTA, pH 8.0, to 100ml of Hanks' salt
solution. Make additions from sterile stock solutions,
maintaining sterility of the final solution. Store at 4°C.
- 70% ethanol: Combine 700ml absolute (not denatured)
ethanol with 300ml water. Store in 0.5-liter
bottles at room temperature.
- Grow cultured HeLa S3 cells in 20 ml of modified
Eagle's α-MEM medium (Invitrogen/Gibco) with 10%
FBS and antibiotics in a 15-cm diameter culture plates
at 37°C with 5% CO2 and 100% humidity (Pai and Bird,
- Collect cells at 60-70% confluence by trypsin
digestion. Harvest just before the rotor is filled with
- Concentrate cells by low-speed centrifugation
(3000g for 5 min) at 4°C and resuspend in 5 ml of ice
cold α-MEM with 5% DHS for every three plates
(15cm). Use this medium throughout the elutriation
procedure. Maintain the cells on ice until they are
loaded into the elutriator.
|FIGURE 2 Assembly and setup of centrifugal elutriator.
(A) Schematic of the centrifugal elutriator. Elutriation medium is
pumped from the reservoir beaker (a) by the peristaltic pump (b)
through the pressure gauge (c) and the sample tube (d) or the bypass
harness (e) through the rotating seal (f) and into the rotor (g). The
sample is separated in the elutriation chamber (h) and is pumped
back to the sample collection tubes on ice (i). (B) Rotor and rotating
seal assembly, including O rings on top of the rotor (j), which seal
the rotating assembly to the rotor, the elutriation chamber (k), and,
from left to right, the outer ring, lower washer, the spring that is
placed inside the lower washer, rotating seal, and top of the seal
assembly (1). (C) Elutriation chamber showing left (outer) and right
(inner) halves separated by the gasket (above). Note the actual separation
chamber within the left side and the set screws and alignment
pins extending beyond both edges of the right side of the
chamber. (D) Strobe assembly located below the rotor. (E) Elutriation
setup showing the reservoir beaker (a), the peristaltic pump (b),
pressure gauge (c), sample tube (d), bypass harness (e), and sample
collection tubes (i). Note the positions of beakers to supply/accept
media. (F) Elutriation centrifuge showing the complete rotating seal
assembly (f), elutriation rotor (g), with strobe wires/waste tubing
extending through the right centrifuge wall, and the inlet/outlet
tubing extending through the left centrifuge wall.
C. Cell Cycle Analysis
- Arrange the elutriation system as described in
the schematic diagram (Fig. 2A). Assemble the elutriator
rotor according to the manufacturer's directions
(Fig. 2B). Lightly lubricate each O ring, which seals the
rotating assembly on top of the rotor, with silicone
grease, taking care to wipe off any excess. Place the
lower washer and spring on top of the rotor followed
by the outer ring and rotating seal. Note that the screw
threads on the top assembly are reversed. Lightly
tighten the top and check that the outer ring spins
freely. Tighten the side set screw and repeat the check
for a freely spinning assembly. Loosen the lower
washer inside the outer ring half a turn. Check that the
assembly spins freely, retighten the lower washer, and
check that it spins freely again. Care should be taken
to ensure that the rotating seal connecting the rotor to
the fluid lines is freshly cleaned and polished with the
scintered glass plate and polishing paper provided.
Only a lint-free tissue with an appropriate solvent
(e.g., CHCl3) should be used as even a small speck of
lint can cause leakage. A very thin layer of silicone
grease can be applied to the upper edge of the rotating
seal to help create and maintain a good seal but all
excess silicone must be removed. Stick the seal to the
polypropylene disk on the bottom surface of the top of the seal assembly by gently seating the silicone on the
seal with a half turn. Carefully screw the top of the seal
assembly onto the outer ring (note reverse threads)
and hand tighten. This connection should not leak
more than 1-3 ml during a normal elutriation run of
<1 h. Even very small leaks encountered at low initial
flow rates can result in significant loss of fluid and cells
toward the end of the run as the flow rate rises. If perpersistent
leaks are encountered, reexamine the seal and
check for a perfectly clean and even surface on both
edges. Repolish the rotating seal if necessary and then
repeat the assembly procedure. If none of these measures
seals the rotor connections adequately, the seal
should be replaced.
- Assemble the elutriator chamber according to the
manufacturer's directions (Fig. 2B). Ensure that both
halves of the chamber are perfectly clean and that the
polypropylene gasket is positioned to allow alignment
of the sample tube (see Section V). Tighten the screws
to assemble but do not overtighten. Lightly lubricate
both O rings, on the base of the chamber assembly,
with silicone grease, taking care to wipe off any excess.
Insert the chamber and align the base pin and sample
tube connections. Secure it in place with the metal
plug. The screw threads on the plug should be lubricated
with Spincoat (Beckman) provided with the
elutriator. Tighten with the tool provided but do not
overtighten as the O rings can be crushed.
- Assemble the elutriator centrifuge according to
the manufacturers directions (Fig. 2C). Remove the
high-speed rotor (if present) from the centrifuge and
wipe out any moisture in the centrifuge chamber.
Install the strobe assembly in the centrifuge chamber
and secure with thumb screws while ensuring that it
is centered over the rotor spindle in the center (Fig.
2D). Feed both wire connections through the ports on
the right side of the centrifuge chamber and secure
them with the metal plate. Carefully place the rotor on
the spindle in the center of the centrifuge and ensure
that it spins freely. Connect the three pieces of silicone
tubing to the rotor (inlet, outlet, and overflow) and
feed them through the appropriate port (inlet and
outlet to the left, overflow to the right with the wires).
Ensure that all are pulled tight enough so that none
have any slack in them but not so tight as to pinch or
pull off any of the connections (Fig. 2F). The overflow
tube should wrap around the top of the seal assembly
and pass under the inlet port (the upper of the two
ports on the sides of the top of the seal assembly). Seal
each of the ports around each tube and wire with a slit
stopper (provided with the centrifuge), where they
pass through the centrifuge chamber wall, to ensure a
- Prepare a large ice bucket containing two 0.5-liter
bottles of elutriation medium, 30 sterile-capped tubes
(50ml), and the cell suspension (Fig. 2E). Place the
bucket on a cart or table next to the centrifuge with the
pump and three 2-liter beakers. One beaker contains 1
liter of sterile water. Include a 20-ml syringe with an
18-gauge needle and the sample injection harness with
a pressure gauge (Cole-Parmer, Cat. No. EW-07380-75).
If sterile samples are to be collected, an additional beaker of 70% ethanol and a bottle of sterile water
must also be included. If elutriated cells are to be cultured
for more than a few hours after separation, a
sterile hood must be positioned next to the centrifuge
to allow both reservoir beakers and samples to be collected
under sterile conditions.
- Clean the exterior of the silicone tubing with
ethanol and place the inlet in the beaker containing the
sterile water and the outlet in an empty beaker. The
inlet tubing should also be installed into the pump
head and attached to the sample application and
bypass harness, with an inline pressure gauge, so that
they are between the pump and the rotor (Fig. 2E).
Begin pumping water through the rotor (which is
stationary and the centrifuge is off, see Section V) at
45ml/min (>200ml). Carefully observe the water as it
fills the equipment (~100ml) and dislodge any bubbles
with gentle pressure on the tubing or tapping of other
components. Release the air from dead spaces within
the pressure gauge by pinching the tubing just after the
gauge and releasing it rapidly. Do not let the pressure
rise above 15psi or the tubing may burst. Adjust the
stopcocks on the sample/bypass harness to allow the
harness, stopcocks, and sample tube to fill completely.
Continue to pump water through the equipment until
all the bubbles have been cleared. Observe all connections,
particularly at the rotor, for leakage.
- Close the centrifuge door and start the rotor.
Allow the speed to gradually rise and stabilize at
2000rpm (±1rpm is acceptable) at 4°C. Be sure to
increase the speed slowly as the fine speed control can
easily overshoot the set point on acceleration. Check
each of the stoppers to ensure that the seals are adequate
to allow development of a vacuum in the centrifuge.
Observe the chamber through the periscope
assembly in the door and adjust the strobe firing timer
to center the image. If the chamber appears to have a
rod running along the center rather than two screws
running near each edge, then the strobe is 180° out of
phase with the rotor and is allowing visualization of
the balance chamber, not the elutriation chamber. Continue
to adjust the strobe until the elutriation chamber
is visible. Check for bubbles at the outlet using the
squeeze-and-release technique described earlier. Turn
off the pump and observe the outlet tubing as you raise
it out of the beaker and suspend it free in the air. If a
slow leak occurs, the fluid and air will be drawn back
up into the tube as the fluid leaks out of the system.
The system is sealed if the fluid level in the outlet tube
does not change.
- If sterile collection is required, switch to 70%
ethanol and pump 200ml through the rotor followed
by 200ml of sterile water from a bottle in the laminar
flow hood. Switch to elutriation medium (without DHS) on ice and pump 200ml (if sterile collection is
not required, omit ethanol rinse). Be sure to include all
sections of the sample injection harness, including
the sample tube and the stopcocks, during the rinse
with each of these solutions. Turn off the pump before
switching the stopcocks. Turn the pump off and
change to elutriation medium with 5 % DHS to prevent
the cells from sticking. Pump 100ml at 45ml/min
through the sample tube. At this point, two people are
required to operate the system: one collects samples
and one monitors rotor and pump speeds as well as
managing sample injection.
- Disperse the trypsinized cells (~1-2 × 108), which
have been held on ice in 5 ml of elutriation medium
(containing 5% DHS), with seven very gentle passes
through an 18-gauge needle on a 20-ml syringe. Be
careful not to introduce bubbles. Turn off the pump.
Gently inject the cells into the sample tube (positioned
with the stopper up), allowing the cells to settle to
the bottom of the tube. Carefully turn the tube over
(stopper down) and gently inject about 2 ml of air into
the sample tube to act as a shock absorber against
the pulsing peristaltic action of the pump. Adjust the
pump, which is still off, to zero and then turn the
pump on. Gradually adjust the pump up to 10 ml/min,
being careful not to overshoot this value. Observe the
sample tube and watch the cells enter the system. Take
care to avoid pulsing of the medium or leaving a residual
pools of cells in the sample tube. Collect three tubes
of 50ml each in the sample collection tubes on ice.
Once the cells are loaded, the harness can be set on the
bench in a stable position that maintains the inverted
orientation of the sample tube (stopper down). Continue
to monitor the entry of the cells into the elutriation
chamber through the periscope. Carefully increase
the pump speed to 15 ml/min. Do not overshoot. We
collect G1-phase cells between 21 and 25ml/min, Sphase
cells at 29-35ml/min, and G2/M-phase cells at
43 ml/min. Be prepared to work quickly at the end of
an elutriation run as the flow rate becomes very fast
and tubes containing elutriated cells fill at the rate of
approximately one every minute. Following elutriation,
pellet the cells by centrifugation (3000g 5min)
and resuspend in growth medium. Adjust cell density
with additional growth medium, after determination
of cell concentration in each sample by counting
in a hemacytometer, and transfer to tissue culture
Part of the synchronous cell fractions are fixed and
analyzed by flow cytometry and the remainder of the
cells are immediately prepared for RNA isolation or placed back into culture for further manipulation (Fig.
3). Cultured cells are analyzed for their ability to synchronously
enter S phase by determining the kinetics
H]thymidine incorporation (Fig. 4).
|FIGURE 3 Flow cytometric analysis of cell cycle synchrony in sequential centrifugal elutriator fractions
of HeLa S3 cells. Synchronous populations of HeLa S3 cells were selected by centrifugal elutriation of exponential
cultures (log), and the degree of synchrony was analyzed by flow cytometry. Cell number was plotted
against DNA content based on propidium iodide fluorescence for each cell cycle fraction. Flow cytometric
analysis of sequential G1-phase fractions collected at flow rates of 21ml/min (G1.1), 23ml/min (G1.2), and
25ml/min (G1.3). S-phase cells were selected by centrifugal elutriation at flow rates of 29ml/min (S0.1) and
35ml/min (S0.2), and G2/M-phase cells were collected at 43 ml/min.
- Phosphate-buffered saline (PBS): Dissolve 0.71 g of
Na2HPO4 (0.01M final, Sigma, Cat. No. S-1934) and
4.5g NaCl (0.9%, w/v final) in ~400ml water and
adjust pH to 7.6. Adjust volume to 500 ml, filter sterilize,
and store at 4°C.
- Staining solution: Make 50µg/ml propidium
iodide (PI) and 40 µg/ml RNase A by dilution of stock
solute/ons. Add 111µl PI stock (4.5mg/ml, Sigma,
Cat. No. P-1764) and 20µl RNase A stock (20mg/ml,
Sigma, Cat. No. R-1859) to 10ml water. Do not attempt
to weigh RNase as any contamination will make future
isolation of RNA very difficult. Open a 250-mg bottle
in the fume hood and add 12.5 ml water. Cap, dissolve,
and boil for 15min to inactivate DNase (Sambrook et al., 1989). Aliquot with a plugged disposable pipette
(tissue culture type) in 1-ml lots in microcentrifuge
tubes. Store at approximately -20°C. Use only disposable
tubes and pipettes with RNase solutions and
avoid any contamination or aerosols. Dissolve 100mg
of PI in 22.22 ml of water directly in the bottle. Do not
weigh out. PI is extremely toxic and is a carcinogen as
well as a potential mutagen and should be handled
with care, including the use of gloves. Be careful not to create aerosols or liberate dust from the granular
reagent. Dispose of all PI solutions and contaminated
materials as hazardous waste. Use only disposable
tubes and pipettes. Store PI at -20°C in the dark as it
is light sensitive.
- 100% trichloroacetic acid: To a 500g bottle of
trichloroacetic acid (TCA, Sigma, Cat.tNo. T-2069), add
sufficient water to bring the volumel in the bottle to
approximately the shoulder. Add a stir bar, cap the
bottle, and stir to dissolve the contents. Carefully
adjust the volume to 500ml. TCA is extremely caustic.
Do not attempt to measure or weigh TCA granules.
Use caution when pipetting the solution. TCA 100%
solution is very stable when stored at approximately
~4°C. Dilutions should be freshly prepared from the
stock daily. Store dilutions on ice while in use and
dispose of unused portions.
- [3H]Thymidine growth medium: Add 10µCi/µl
[3H]thymidine (1 µCi/µl stock, PerkinElmer Life and
Analytical Sciences, Inc., Cat. No. NET-027Z) directly
to growth medium under sterile conditions at the rate
of 10 µl/ml of medium. Prepare fresh on a daily basis.
- 1.0M Tris buffer: Dissolve 60.55g Tris buffer
(Fisher, Cat. No. BP152-1) in ~400ml water and adjust
pH to 7.6. Adjust volume to 500 ml, filter sterilize, and
store at 4°C.
- 20% sodium dodecyl sulfate (SDS): Dissolve 20g SDS (Sigma, Cat. No. L-1926) in sterile water using a
sterile beaker and a stir bar rinsed in ethanol. Adjust final volume to 100ml. SDS cannot be autoclaved or
filtered. Store at room temperature.
- TES buffer: Add 10mM Tris-HCl, pH 7.6 (1 ml of
1M stock), 1 mM EDTA, pH 8.0 (0.5 ml of 0.5 M stock),
and 1% SDS (5 ml of 20% stock) to 93.5 ml water, filter,
and store at room temperature.
- 4% paraformaldehyde: Dissolve 4 g paraformaldehyde
(Electron Microscopy Sciences, Cat. No. 15710) in
100ml water and stir to dissolve by heating gently to
60-65°C in a fume hood. This can take an extended
period and the solution will still appear cloudy. Clarify
by the addition of a few drops of 1M NaOH (up to ~20
drops). Cool before use and store at 4°C. Fixative
fumes are extremely toxic. Always use in a fume hood
and avoid any contact.
|FIGURE 4 Analysis of synchrony of entry into DNA
centrifugal elutriation fractions of HeLa S3
cells. HeLa cells were
separated into synchronous
fractions by centrifugal elutriation, and
kinetics of entry
into DNA synthesis phase (S phase) was determined
by measuring mean acid-precipitable [3H]thymidine
(10µCi/ml in growth medium) for five
incubations, in duplicate, for each cell cycle
fraction identified in Fig.
3. Note that each cell fraction
reaches S phase at sequentially later
times after return to
A. Theoretical Basis of Separation
- Analyze cell cycle fractions by flow cytometric
analysis (Fig. 3). Wash approximately 5 × 105 cells by centrifugation and resuspend in ice-cold Hanks' balanced
salt solution. Fix cells by addition of an equal
volume of ice-cold 4% paraformaldehyde. After a 24-
h incubation at 4°C collect the cells by centrifugation
and resuspend in 2 ml of ice-cold PBS. Alternatively, fix
cells by slow dropwise addition of 3 volumes of 70%
ethanol (-20°C while applying continuous gentle agitation
with a vortex mixer. Allow cells to fix for at least
1 h at 4°C and then wash as described earlier. Approximately
30min prior to flow cytometric analysis, add
3ml of staining solution to each aliquot of 300µl of
cell suspension and incubate at room temperature.
Analyze fluorescence on a flow cytometer (Elite Flow
Cytometer, Coulter Electronics).
- Analyze acid precipitable [3H]thymidine incorporation
by synchronous cell populations by pulse
labeling each separated fraction of cells with 10 µCi/ml
[3H]thymidine in complete growth medium (Fig. 4) for
1 h at hourly intervals after return of the cells to culture
(Wu et al., 1993; Bird et al., 1988). Place cells in 96-well
plates (2 × 104/well) in 100µl growth medium and
incubate under normal growth conditions. Add 1 µCi
[3H]thymidine to each well at the appropriate time and
incubate for 1 h at 37°C. Wash each cell fraction twice
with Hanks' balanced salt solution, drain fluid, lyse in
100µl TES, load 100btl of lysate onto a 2.4-cm filter
paper circle (Whatman 540, Cat. No. 1540-324) labeled
with a No. 2 pencil, and allow to air dry. Precipitate
samples with excess solutions of 200ml for up to 50
filters for 20min (do not exceed this time or the filters
may be damaged): 20% TCA, 10% TCA, absolute
ethanol, ether, absolute ethanol. Then, air dry and
determine the radioactivity in each sample by liquid
scintillation counting in a 5-ml ScintiSafe Econ 1 scintillation
fluor (Fisher, Cat. No. SX20-5) as described
previously (Wu et al., 1993; Bird et al., 1988).
Cells are separated in the centrifugal elutriator by a
process that was originally called counterstream centrifugation.
This process is based on the opposition of
two forces: media flow and angular acceleration due
to an applied centripetal force. All of the cells in the
suspension to be separated by centrifugal elutriation
are pumped under gentle pressure into the separation
chamber, entering at the extreme outside end of the
chamber where the radius of rotation is maximal (rmax
to begin accumulating in the chamber (Fig. 1). As the
cells fill the chamber, they change direction (as the loading tube turns) from flowing out to the perimeter
of the rotor to flowing toward the center of the rotor.
As they do, the particles enter a chamber that is
designed with a conical profile. As the particles enter
the chamber, the cross-sectional area increases rapidly,
dramatically decreasing inward movement of the cells
under a constant flow rate. Thus, the cells begin to
decelerate as the cross-sectional area of the chamber
increases. At some point, determined by cell size,
media flow rate and viscosity, and centripetal force, the
cells cease to move inward and become suspended in
the chamber at a position where cell movement inward
due to flow rate is balanced by apparent centrifugal
force outward. At this point, medium is flowing by the
cells but inward movement of the cells is offset by the
apparent outward centrifugal force applied due to the
angular momentum of the rotor. The population of
cells has thus reached equilibrium and their position
remains unchanged, although the cells tend to sort
themselves under this combination of opposing forces
with the smallest particles sorting closest to the center
of the rotor.
There are now two choices for the operator with
respect to fractionation of the cells: rotor speed can
be decreased incrementally or the flow rate can be
increased incrementally. In practice, the latter choice is
the more practical due to the ease and precision with
which the pump can be adjusted compared to the centrifuge.
In either case, adjustments are made such that
the equilibrium position of the smallest population of
cells can move inward into the chamber until it reaches
the constriction in the chamber where flow rate accelerates
due to a rapid decrease in cross-sectional area.
Thus, by adjusting the flow rate up in small increments
(size of the increments must be determined empirically),
cells can be fractionally separated from the
The actual elutriation characteristics depend on the
shape and size of the cells in the population. For
trypsinized cells, whose specific gravity is not much
greater than the buffer in which they are suspended,
shape generally approaches spherical unless cell processes
or applied forces alter this. Thus, separation is
usually the product of apparent cell diameter. Centrifugal
elutriation has been shown to be unusually
subtle in its ability to discriminate subfractions based
on very small differences in cell size. For example, in
separation of cells in different phases of the cell cycle,
cells approximately double in volume as they pass
through the cell cycle from G1
phase to mitosis
(Mitchison, 1971). This translates into an increase of
only about 26% in the radius of an average premitotic
cell compared to an average freshly divided G1
cell. This relationship can be demonstrated by the following equations where V is cell volume and r is cell
radius, assuming a spherical shape. The G1
is represented by a volume of VG1
and an apparent
radius of rG1
, the premitotic cell is represented by a
volume of VM and an apparent radius of rM
, and the
assumption is made that cell volume exactly doubles
on average during the intervening period.
|4/3 π (rG1)3 = VG1
|1 (volume of a spherical G1 phase cell)
|4/3 π (rG1)3 = VM
|2 (volume of a spherical premitotic cell)
|2 VG1 = VM
|3 (volume doubles during the cell cycle)
|Substituting Eqs. (1) and (2) into Eq. (3) for each
volume and then simplifying,
|2[4/3 π (rG1)3] = [4/3 π (rM)3]
|2 (rG1)3 = (rM)3
Taking the cube root of each side,
|1.26 rG1 = rM
Thus, the radius of the largest cell in the cell cycle,
on average, has an apparent radius (and diameter)
only 26% larger than the smallest cell on average. We
have successfully separated cells in this narrow continuum
of cell sizes into seven distinct fractions with
little overlap. This demonstrates that the subtle differences
in cell size that occur as cells proceed through
the cell cycle can be detected and efficiently separated
using this technique. Successful separation was based
on only about a 3-5% difference in radius (or diameter)
between succeeding fractions that could be discriminated
clearly by centrifugal elutriation.
The theoretical basis on which centrifugal elutriation
is capable of separating cells, by balancing changes
in medium flow balanced against angular momentum,
has revealed that sequential fractions were frequently
differentiated by as little as a 3% difference in cell
diameter. In most cases, apart from the expense of purchase
of the elutriation centrifuge, the only compromise
consists of a somewhat lower level of purity in
some samples, although purities approaching 100%
have been reported. To balance this drawback, great
improvements in yield, speed, and gentleness of
handling of the cells have been achieved. Due to
these obvious benefits, centrifugal elutriation has been
applied to a wide variety of cells and cell types to successfully
affect separation based on cell size.
The theoretical considerations described earlier
have been applied to allow separation of a great variety of cell types and separation of cultured cells
based on cell cycle phase (reviewed extensively in
Conkie, 1985; Beckman Instruments, 1990). Using this
protocol, it is possible to separate approximately 1× 108
to 1 × 109
cells into up to eight sequential cell
cycle phase fractions in about 40-60min (sequential
fractions designated: G1.1
, see Figs. 3 and 4). Later cell cycle fractions
contained increasing numbers of contaminating cells
from the phase preceding it. The first fractions were of
the highest purity (approximately 97%, see Fig. 3)
while the purity of the fractions dropped (to approximately
90%) in samples representing later times
during the cell cycle. In G2
/M fractions, significant
numbers of cells synthesizing DNA are recovered,
although this activity declines rapidly once the cells
are placed back in culture (Fig. 4). Only centrifugal elutriation
can produce so many fractions of relatively
high purity and of such large size so quickly with
little detectable lag in cell growth and without
- In the time since we acquired our centrifugal
elutriator, Beckman Instruments has released newer
models designed to separate, among other qualities,
larger numbers of cells. While the principles governing
separation remain the same, details of procedures
necessary in setting up the rotor and the seals may
differ, depending on the model, and thus the manufacturer's
directions should be adhered to carefully to
ensure correct assembly and operation.
- It is critical that separations be attempted
only with single cell populations. Steps should be
taken to ensure complete dissociation of cells
liberated from tissues or culture vessels. When in
doubt, dispersion of samples should be monitored
- Only Beckman neutral pH rotor detergent should
be used to clean the rotor and separation chamber. It
is particularly important to ensure that the sample
tube at the outer edge of the separation chamber is
soaked in detergent overnight to remove any cell
debris that has accumulated as this will affect flow rate
greatly as well as the fluid dynamics of sample loading
in the chamber. This aperture is too small to be cleaned
with a tool, and no instrument should be applied that
could scratch the interior surface of the chamber. The
operator must be extremely careful with the separation
chamber, as overtightening of the screws or scratches to the surface will damage the chamber, affecting performance
- If alterations to the growth medium or elutriation
medium are contemplated, the new medium must be
tested to empirically determine at what flow rate cell
cycle fractions elute. Even very small changes in media
formulation (e.g., as little as 0.5% change in DHS concentration)
will change the fluid dynamics of the elutriation
system dramatically. Changes in cell-loading
density and temperature can also create detectable
effects on elutriation profiles. A simple pilot experiment
is usually sufficient to determine what effects
such alterations have on elution flow rate if it is followed
by flow cytometric analysis.
- All O rings and gaskets should be inspected
before each run to ensure that they are in good condition.
All worn seals should be replaced.
- Failure to secure the wires and tubing connecting
the rotor and strobe light to the exterior of the centrifuge
will result in them becoming wrapped around
the rotor, resulting in serious damage to the
- We have replaced the Oakridge-style sample
application tube supplied by the manufacturer with a
straight glass test tube (13 × 100mm) with the same
size aperture at the top as the tube supplied. This eliminates
the shoulder at the top of the Oakridge tube,
which can trap cells, resulting in a continuous residual
loading of cells throughout the elutriation procedure.
The tube should be siliconized to within approximately
2cm from the top with Gel Slick (FMC Bio-
Products). Gel Slick should not be allowed to contact
the glass surface above this point as the stopper will
no longer hold securely under pressure.
- Rotor speed should be monitored frequently
during the run as fluctuations of only a small amount
can affect purity of the samples greatly. It is particularly
important to check rotor speed each time that the
refrigeration system in the centrifuge cycles on.
- If sterilization of the assembly is required, be
sure that the centrifuge is turned off while the ethanol
is in the system. Failure to observe this safety measure
could result in a fire hazard. Alternatively, the system
can be sterilized with 6% hydrogen peroxide while the
centrifuge is running (Conkie, 1985).
The author thanks Dr. Gin Wu, Dr. Suresh Pai, and
Dr. Shiawhwa Su for consultation on this protocol
and Patricia DeInnocentes and Randy Young-White
for valuable technical support. The author also
thanks Dr. Lauren G. Wolfe for critical reading of the
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