Download Isolation of Monocyte/Macrophage Populations

UNIT 7.6
Isolation of Monocyte/Macrophage Populations
The following basic and alternate protocols describe the isolation of monocytes from
lymphocytes by adherence, gradient sedimentation on colloidal silica particles, and flow
cytometry. Because the first two methods can result in cell activation (induction of gene
expression or protein secretion), and the third is technically difficult, a fourth protocol is
presented which describes counterflow centrifugal elutriation. This latter procedure can
be used to isolate large numbers of purified, nonactivated monocytes. Flow cytometry
methods for measuring the expression of membrane determinants to characterize the
phenotypic profiles of monocytes and macrophages can be found in UNIT 14.3; functional
assays using isolated monocytes/macrophages (e.g., assays of oxidative metabolism,
phagocytosis, and anti-viral and anti-tumor activities) are also covered in Chapter 14.
CAUTION: When working with human blood, cells, or infectious agents, biosafety
practices must be followed (see Chapter 7 introduction).
NOTE: All solutions and equipment coming into contact with cells must be sterile, and
proper sterile technique must be used accordingly.
BASIC
PROTOCOL
ISOLATION OF MONOCYTES BY ADHERENCE
In this protocol, monocytes are isolated from mononuclear cells by exploiting their ability
to adhere to glass or plastic. This is a quick and easy procedure, but it can induce cell
activation (see background information).
Materials
Peripheral blood mononuclear cells (PBMC; UNIT 7.1)
Serum-free DMEM (e.g., GIBCO/BRL #320-1960), supplemented with 2 mM
L-glutamine and 50 µg/ml gentamycin
0.02% EDTA in PBS (APPENDIX 2; low pyrogen and without Ca++ or Mg++)
75-cm2 tissue culture flasks (Falcon #3023)
Beckman GPR centrifuge with GH-3.7 horizontal rotor (or equivalent)
15-ml conical polypropylene centrifuge tube
Additional reagents and equipment for cell counting (APPENDIX 3)
1. Suspend PBMC in supplemented serum-free DMEM at 2 × 106 cells/ml.
2. Add 10 ml of the cell suspension to each 75-cm2 flask and incubate 1 hr in a humidified
37°C, 5% CO2 incubator.
Some investigators add 10% autologous human serum (Edelson and Cohn, 1976) or 10%
pooled human serum (Gartner et al., 1986) to DMEM during the initial adherence step.
Although cells adhere in the presence of serum, they adhere more quickly in its absence.
3. Decant the medium which contains nonadherent cells, wash twice with 10 ml
supplemented serum-free DMEM to remove any residual nonadherent cells, and
replace with 10 ml fresh DMEM.
4. Remove adherent monocytes by gently scraping with a plastic cell scraper or by
incubating the cells in ice-cold 0.02% EDTA/PBS solution for 10 min followed by
firmly tapping the flask. Transfer cells to a 15-ml conical tube and centrifuge 10 min
in GH-3.7 rotor at 1400 rpm (300 × g) to remove the EDTA/PBS solution.
Isolation of
Monocyte/
Macrophage
Populations
5. Resuspend in supplemented serum-free DMEM and count monocytes.
Alternatively, monocytes can be adhered in the appropriate culture plate and used directly.
7.6.1
Supplement 16 CPI
Copyright © 1995 by John Wiley & Sons, Inc.
ISOLATION OF MONOCYTES BY SIZE SEDIMENTATION
In this procedure, monocytes are isolated from cell suspensions by size sedimentation
using a continuous gradient of colloidal silica particles (Gmelig-Meyling and Waldmann,
1980). This avoids the adherence step of the previous protocol. Nevertheless, activation
of monocytes is still possible (see background information).
BASIC
PROTOCOL
Materials
Fetal calf serum (FCS; heat-inactivated 1 hr, 56°C)
Peripheral blood mononuclear cells (PBMC; UNIT 7.1)
Hanks balanced salt solution (HBSS; APPENDIX 2) containing 10% FCS
Percoll (specific gravity 1.130 g/ml, 17 mosM/kg; Pharmacia #17-0891-01)
2× PBS (GIBCO/BRL #310-4200AJ or APPENDIX 2)
Sorvall RC2-B centrifuge with SS-34 fixed-angle rotor (or equivalent)
Beckman GPR centrifuge with GH-3.7 horizontal rotor (or equivalent
temperature-controlled centrifuge)
15-ml conical polypropylene centrifuge tube
15-ml polycarbonate centrifuge tube (Sorvall #00770)
1. Place 3 ml FCS in a 15-ml centrifuge tube and carefully overlay with PBMC.
Centrifuge 15 min in a GH-3.7 rotor at 800 rpm (200 × g), 18° to 20°C. Discard
supernatant, which contains platelets. Resuspend pellet in HBSS/FCS at 2-5 × 107
cells per 1 to 2 ml.
2. In a 15-ml polycarbonate centrifuge tube, add 7 ml Percoll to 6 ml of 2× PBS.
Centrifuge 40 min in Sorvall SS-34 rotor at 15,000 rpm (21,000 × g), room temperature, to form the continuous gradient.
3. Gently layer the platelet-free mononuclear cells from step 1 onto preformed gradient.
4. Centrifuge 20 min in GH-3.7 rotor at 2400 rpm (1000 × g), 4°C.
5. Using sterile Pasteur pipets, carefully collect the four opaque bands of cells beginning
at the top. Band 1 contains dead cells, debris, and a few platelets. Band 2 contains
monocytes, a few lymphocytes, and any remaining platelets. Band 3 contains lymphocytes, and a few monocytes. Band 4 contains granulocytes and RBC.
6. Wash and count the cells in band 2 (APPENDIX 3).
Monocytes comprise 70% to 90% of band 2.
ISOLATION OF MONOCYTES BY FLOW CYTOMETRY
In this protocol, monocytes are separated from mononuclear cell populations by flow
cytometry. Although minimal activation is caused by this method, the flow cytometer can
be difficult to use.
Materials
Peripheral blood mononuclear cells (PBMC; UNIT 7.1)
PBS (APPENDIX 2), without and with 10% AB serum (heat-inactivated 1 hr, 56°C)
Serum/azide solution: 0.1% NaN3 and 2% FCS in PBS
Azide solution: 0.1% NaN3 in PBS
10 µg/ml monocyte-specific monoclonal antibody in serum/azide solution
(e.g., Leu M3 or Leu M5; Becton Dickinson)
Serum-free DMEM (e.g., GIBCO/BRL #320-1960), supplemented with
2 mM L-glutamine and 50 µg/ml gentamycin
Beckman GPR centrifuge with GH-3.7 horizontal rotor (or equivalent)
BASIC
PROTOCOL
Immunologic
Studies in
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7.6.2
Current Protocols in Immunology
Additional reagents and equipment for immunofluorescent staining (UNIT 5.3) and
flow cytometry (UNIT 5.4 & 7.9)
1. Suspend PBMC in PBS and centrifuge 10 min in a GH-3.7 rotor at 1400 rpm (300 ×
g), 4°C. Aspirate and discard the supernatant.
2. Resuspend the pellet in PBS containing 10% AB serum and incubate 10 min at 4°C.
Centrifuge 10 min at 300 × g, 4°C, to pellet cells.
This step blocks nonspecific binding of MAb to surface Fc receptors.
3. Resuspend pellet at 1.0-1.5 × 107 cells/ml in serum/azide solution containing 10
µg/ml monocyte-specific MAb. Incubate 20 min at 4°C.
If the MAb reacts with surface determinants on other cells, the purity of the sorted cells
will be reduced. Leu M3 and Leu M5 are monocyte-specific and have low cross-reactivity
with other cell types. In addition, they do not cause monocyte activation.
4. Centrifuge cells 10 min at 300 × g, 4°C. Wash cells by resuspending in 3 ml azide
solution, and centrifuging 3 min at 300 × g, 4°C.
5. Resuspend the cells in supplemented serum-free DMEM at ≥1 × 106 cells/ml.
6. Sort by flow cytometry.
7. Resuspend monocytes in supplemented serum-free DMEM and keep on ice prior to
use.
BASIC
PROTOCOL
ISOLATION OF MONOCYTES BY COUNTERFLOW
CENTRIFUGAL ELUTRIATION
Counterflow centrifugal elutriation (CCE) is a highly effective method for separating
large numbers of monocytes from Ficoll-Hypaque purified PBMC (UNIT 7.1). The protocol
has four parts: assembly of the CCE system, preparation of mononuclear cells for CCE,
loading of mononuclear cells, and isolation of monocytes by CCE. In contrast to the
adherence and sedimentation methods of isolation, this procedure does not lead to
monocyte activation.
Materials
70% ethanol
Phosphate-buffered saline (PBS; APPENDIX 2) low pyrogen and without Ca++ and
Mg++
Peripheral blood mononuclear cells (PBMC; UNIT 7.1)
Isolation of
Monocyte/
Macrophage
Populations
Beckman elutriation system: J-6M elutriation centrifuge with JE-6B rotor, strobe,
and standard elutriation chamber(s)
Tissue culture hood (biosafety cabinet)
2-ml pipet
Silastic tubing, 1⁄4 × 118–in.
Masterflex pump (1 to 100 rpm, 10-turn potentiometer, 115 VAC with 7014 pump
head, Cole-Parmer #N-07520-35 and silicone tubing, #F6411-14, respectively)
Ring stand and holder
Beckman GPR centrifuge with GH-3.7 horizontal rotor (or equivalent)
Coulter counter with channelyzer
10-ml pipet or 10-ml syringe
50-ml polypropylene centrifuge tubes
Additional reagents and equipment for Ficoll-Hypaque cell separation (UNIT 7.4),
flow cytometry (UNIT 5.4), and nonspecific esterase staining (APPENDIX 3)
7.6.3
Current Protocols in Immunology
Assemble the elutriation system
1. Assemble the elutriation rotor and the strobe setup according to the manufacturer’s
instructions. Before attaching the seal housing, be sure that the spring forces the
rotating seal against the seal housing by pressing the rotating seal down on the shaft.
The spring should force the rotating seal back up when the pressure is released.
2. Assemble the inlet line from a tissue culture hood to the rotor in the centrifuge as
shown in Figure 7.6.1. Connect a 2-ml pipet to silastic tubing and attach this tubing
to the silicone tubing on the inlet side of the Masterflex pump. Assemble the sample
reservoir using silastic tubing as shown in Figure 7.6.2A. Connect the silicone tubing
on the outlet side of the pump to the silastic tubing on the sample reservoir setup as
shown in Figure 7.6.1.
The setup for the sample reservoir (Fig. 7.6.2) differs from that suggested by the manufacturer. An additional three-way valve has been added, and the position of the needles is
reversed. This allows the sample chamber to be used as a bubble trap and a pulse dampener
during elutriation and, as discussed below, as a mechanism to slowly introduce the cells
into the elutriation chamber(s) at a controlled flow rate. A 70-ml sample chamber is shown
in this setup; however, a 30-ml chamber can be used in the same manner. Also note that
the pressure indicator supplied with this equipment (to detect pressure buildup as a
result of air bubbles) has been eliminated due to contamination that can occur in its
housing. Problems with back pressure can be eliminated by ensuring that there are no
bubbles in the system (see below) and by using the sample reservoir as a bubble trap
during the run.
3. Connect the silastic outlet line from the rotor to the silicone tubing as described by
the manufacturer. Route this tubing back to the tissue culture hood, connect it to a
2-ml pipet with a short piece of silastic tubing, and attach the pipet to a holder on a
ring stand (Fig. 7.6.1).
4. To sterilize the elutriation system, place the pipet on the inlet line into a beaker
containing 300 ml of 70% ethanol and turn the pump on at flow rate of 5 to 8 ml/min.
(Use the settings on the 10-turn potentiometer of the pump corresponding to flow
rates between 5 and 19 ml/min.) Turn the three-way valves to positions that will
initially bypass the sample reservoir. Once the ethanol has gone through the system
and is being collected in a beaker under the pipet connected to the outlet line, manually
spin the rotor clockwise until all of the air in the chambers in the rotor has been
removed. Remove the sample reservoir, leave the needles connected to the tubing,
Culture hood
J-6M elutriation
centrifuge
silicone
silastic tubing
tubing
sample
reservoir
setup silicone
tubing
pump
silastic
tubing
10turn
potentiometer
1 liter
of PBS
ice
elutriation
JE-6B
chamber elutriator rotor
Figure 7.6.1 Design of counterflow centrifugal elutriation system.
Immunologic
Studies in
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7.6.4
Current Protocols in Immunology
and run 70% ethanol through each side of the setup by opening and closing the
appropriate three-way valves. Then turn the three-way valves so that the sample setup
is by-passed again, and continue to pump the remaining ethanol through the system.
5. After the ethanol wash, place the inlet pipet in PBS, and pump 400 ml through the
elutriation system following the protocol outlined for ethanol in step 4. Rinse the
sample chamber with 70% ethanol and then with PBS, and reattach it to the sample
chamber setup.
Prepare mononuclear cells for elutriation
6. Isolate PBMC by density-gradient sedimentation on Ficoll-Hypaque.
7. Resuspend the PBMC in PBS and centrifuge 10 min in a GH-3.7 rotor at 800 rpm
(200 × g), room temperature. This should leave most remaining platelets in suspension while pelleting the mononuclear cells. Decant the supernatant and resuspend
mononuclear cells in 30 ml PBS.
8. Determine the approximate number of monocytes by selectively counting the cells
on the Coulter counter channelyzer, which provides a profile of the cell size that
allows for discrimination between lymphocytes (first peak) and monocytes (second
peak).
A maximum of 1.5 × 109 mononuclear cells are retained in the standard separation
chamber for the JE-6B rotor. When mononuclear cells are obtained from leukapheresis,
the number of monocytes may be 2-3 × 109. To purify this many monocytes, put a second
standard separation chamber in the rotor in place of the by-pass chamber. Mount this
chamber with the indicator pin on the top of the shelf in the rotor rather than in the
hole designated for the pin. This is necessary because the lines on the bypass side
of the JE-6B rotor are reversed. Also, be sure to use a flow rate in the elutriation
described below that will elute most of the lymphocytes while retaining the monocytes.
Load the mononuclear cells
9. Turn the knobs up on the three-way valves located on the inlet and outlet needle lines
of the sample reservoir setup (Fig. 7.6.2A). Empty the sample reservoir with either
a pipet or syringe attached to the injection port. Using a 10-ml pipet or syringe, load
A
T connector
to rotor
injection port
pipet
flow
Luer lock connector
from
pump
B
to rotor
flow
from
pump
3-way valves
Luer lock
connectors
70 ml sample
reservoir
Isolation of
Monocyte/
Macrophage
Populations
Figure 7.6.2 Configuration of sample reservoir during loading of cells (A) and during elutriation (B).
7.6.5
Current Protocols in Immunology
the cells into the sample reservoir through the three-way valve attached to the line
with the long needle (Fig. 7.6.2). Confirm that there is no air in the injection line by
filling the line with PBS. Before removing the pipet or syringe, turn the knob on the
three-way value 90° to close the injection port. Also turn the knob on the opposite
three-way valve 90° (the air in this line will be trapped in the sample chamber when
the elutriation procedure starts).
10. Set the J-6M centrifuge to 1960 rpm (500 × g), 18°C, and the top three-way valve on
the sample reservoir setup so that the flow will be directed through the short needle
into the chamber (Fig. 7.6.2B).
11. Set the pump flow rate to 10 ml/min; this will result in the lymphocytes being eluted
or forced out of the separation chamber(s) in the rotor while the monocytes are
retained. Pump 300 ml PBS through the system in order to load all the cells in the
sample reservoir as well as elute most of the lymphocytes from the separation
chamber(s).
Swirl the sample reservoir occasionally to prevent cells from accumulating on the bottom
of the tube. Also, monitor the size of the cells exiting from the elutriator on the Coulter
counter channelyzer to ensure that only lymphocytes are being eluted.
Isolate the monocytes
12. Gradually increase the flow rate at increments of 0.5 ml/min, and pump 100 to 150
ml PBS through the system for each increment. Closely monitor the exiting cells on
the Coulter counter.
13. When the ratio of monocytes to lymphocytes exiting the elutriator exceeds 50%
(usually ∼11.5 to 12 ml/min), increase the flow rate to 19 ml/min and collect the
exiting monocytes in six 50-ml tubes.
14. Turn off the centrifuge and wash out remaining cells or clumps of cells with PBS.
15. Centrifuge cells 10 min at 800 rpm (150 × g), room temperature, and resuspend the
cells in DMEM. Determine the number of monocytes on the Coulter counter. The
purity can be further assessed by flow cytometry or nonspecific esterase staining.
After completing elutriation, pump distilled water and then air through the system.
Occasionally, pump 50% chlorine bleach solution through the system to remove
accumulated protein. Disassemble the rotor after each elutriation to minimize corrosion and rust.
COMMENTARY
Background Information
Monocytes and tissue macrophages (collectively referred to as mononuclear phagocytes)
play a critical role in host immune responses
through their ability to act as phagocytes, to
serve as accessory cells for mitogen-and antigen-specific lymphocyte responses, and to secrete soluble products (monokines). Thus, the
isolation and study of this cell population is
important to the understanding of host defense
and the pathogenesis of inflammation.
Monocytes are released from the bone marrow into the blood where they have a half-life
of ∼8 to 9 hr. Isolation of these cells has the
potential of allowing study of mononuclear
phagocytic cells prior to their activation. The
circulating monocytes migrate to inflammatory
sites and tissues where they differentiate into
long-lived resident macrophages. Isolation of
these cells requires initial dispersal of the tissue
under investigation, sometimes using collagenase and other agents. The cells isolated are
more mature cells of the mononuclear phagocyte family that have frequently undergone
activation in vivo.
Following the initial separation of mononuclear cells (monocytes and lymphocytes) from
the starting cell population (UNIT 7.1), monocytes
are separated from lymphocytes. In the first
three procedures described in this unit—adher-
Immunologic
Studies in
Humans
7.6.6
Current Protocols in Immunology
ence, size sedimentation, and flow cytometry—
this is accomplished on the basis of the following properties of monocytes: their ability to
adhere to glass or plastic, their unique size and
density compared with other lymphoid cells,
and their ability to express specific surface
antigens.
The advantage of separating monocytes by
adherence is that the procedure is quick, easy,
and does not require complicated equipment.
However, adherence is an activation event that
can induce both gene expression and protein
secretion (Fuhlbrigge et al., 1987; Haskill et al.,
1988). Separation by sedimentation with colloidal silica particles could potentially activate
monocytes since silica has been shown to activate monocytes (Heppelston and Styles, 1967).
Cell sorting by flow cytometry induces minimal cell activation using appropriate antibodies
that do not induce signal transduction; however,
this procedure results in relatively low cell
yields. To circumvent the problems of activation and low yields, isolation of monocytes by
counterflow centrifugal elutriation (CCE) is
recommended. This procedure is particularly
appropriate for isolating monocytes for gene
expression studies.
CCE separates cells on the basis of size
and density. The cells are loaded in a separation chamber within a centrifuge rotor in a
flow pattern which runs against, or counter
to, the centrifugal force of the spinning rotor.
Thus, the flow rate counters the force of
gravity (g) causing the cells to stratify in the
chamber in a zone where the sedimentation
rate of the cell is balanced by the flow rate.
Accordingly, the smaller (less heavy) cells
are closer to the top of the chamber or the
center of the centrifuge. These cells are the
first to exit the separation chamber(s) and
rotor as the flow rate is increased. By taking
advantage of the differing sizes of the
mononuclear subsets, the B cells, T cells, and
monocytes, in that order, can be separated by
CCE (Wahl et al., 1984).
Critical Parameters
and Troubleshooting
Isolation of
Monocyte/
Macrophage
Populations
Two problems occur using the first three
methods for isolating monocytes. The isolated
monocyte population may be contaminated by
2% to 10% non-monocytic cells. This contamination may be higher when isolating monocytes
by sedimentation from patients with leukemia,
because abnormal lymphoblasts may sediment
with the monocytes. Also, the number of monocytes isolated is limited by the amount of sur-
face available for adherence, the number of
cells that can be layered onto the gradient, and
the sorting capacity of the flow cytometer.
Thus, only 60% to 90% of the starting monocytes may be recovered, depending on the procedure used.
In the CCE procedure, air in the system can
be a major problem. Air bubbles in the lines and
the rotor must be eliminated before elutriation
begins. While pumping ethanol or PBS through
the system, air bubbles can be forced out of the
rotor by manually spinning the rotor, and/or by
intermittently pinching the inlet or outlet lines
from the rotor.
Another potential problem with CCE is cells
exiting from the rotor before being separated.
They will have the appearance of the unfractionated population. This is usually caused by
mixing of the inlet and outlet flow at the surface
of the rotating seal. Therefore, it is crucial that
the rotating seal makes contact with the seal
housing, as described in the procedure, and that
the O-ring on the top of the shaft be inspected.
This O-ring is very prone to damage and should
be inspected each time the rotor is assembled.
If there is leakage around the rotor, the chambers should also be inspected to be sure that
they are assembled correctly and that the Orings are in place.
Anticipated Results
The purity of the monocyte population usually exceeds 90%, with 60% to 90% of the
starting cells recovered in the first three protocols. In CCE, monocyte recovery depends on
mononuclear cell density in the chamber. With
up to 1.5 × 109 total mononuclear cells, yields
of 75% to 95% are obtained. In contrast, with
5-9 × 109 mononuclear cells (containing 1-1.5
× 109 monocytes), recoveries are likely to be
lower (50% to 80%), because the large number
of lymphocytes that are exiting the rotor carry
some of the monocytes with them. At all concentrations, however, purity of the isolated
monocytes is 96%.
Time Considerations
The quickest method for isolating monocytes is adherence, which takes 1 hr. Isolation
by colloidal silica particle gradient sedimentation takes ∼2 hr. Isolation by flow cytometry
takes 3 to 5 hr, depending on the purity and size
of the starting population and the desired purity
and number of cells in the sorted population.
Routine assembly of the elutriation system,
once the initial tubing is in place, takes 15 to
20 min. Approximately 60 to 80 min are need-
7.6.7
Current Protocols in Immunology
ed for preparation of the cells. The elutriation
procedure requires ∼90 min. Thus, the entire
procedure can be completed in ∼3 hr.
Haskill, S., Johnson, C., Eierman, D., Becker, S. and
Warren, K. 1988. Adherence induces selective
mRNA expression of monocyte mediators and
proto-oncogenes. J. Immunol. 140:1690-1694.
Literature Cited
Heppelston, A.G. and Styles, J.A. 1967. Activity of
a macrophage factor in collagen formation by
silica. Nature 214:521-522.
Edelson, P.J. and Cohn, Z.A. 1976. Purification and
cultivation of monocytes and macrophages. In In
Vitro Methods in Cell-Mediated and Tumor Immunity (B.A. Bloom and J.R. David, eds.) pp.
333-340. Academic Press, San Diego.
Fuhlbrigge, R.C., Chaplin, D.D., Kiely, J.M., and
Unanue, E.R. 1987. Regulation of interleukin 1
gene expression by adherence and lipopolysaccharide. J. Immunol. 138:3799-3802.
Gartner, S., Markovitz, P., Markovitz, D.M., Kaplan,
M.H., Gallo, R.C. and Popovic, M. 1986. The
role of mononuclear phagocytes in HTLVIII/LAV infection. Science 233:215-219.
Gmelig-Meyling, F. and Waldmann, T.A. 1980.
Separation of human blood monocytes and lymphocytes on a continuous percoll gradient. J.
Immunol. Methods 33:1-9.
Wahl, L.M., Katona, I.M., Wilder, R.L., Winter,
C.C., Haraoui, B., Scher, I. and Wahl, S.M. 1984.
Isolation of human mononuclear cell subsets by
counterflow centrifugal elutriation (CCE). I.
Characterization of B-lymphocyte-, T-lymphocyte-, and monocyte-enriched fractions. Cell.
Immunol. 85: 373-383.
Contributed by Larry M. Wahl
and Phillip D. Smith
National Institute of Dental Research/NIH
Bethesda, Maryland
Immunologic
Studies in
Humans
7.6.8
Current Protocols in Immunology
Supplement 17