- Email: [email protected]
[4] Agarose method for human neutrophil chemotaxis
50
CHEMOTAXIS
[4]
This is the figure we would expect on the basis of random migration alone, and the figures in parentheses in Table I are based on similar calculations (but with more points, i.e., Vo, V1, V2, 1/3, etc. and dl, dE, d3, etc. (based on the figures on the diagonals). If, in an actual experiment, the cells had moved 95/zm/hr instead of 68/tm in a linear gradient from 0 to 10/zg/ml, this would be much higher than would be expected from random locomotion alone, and would indicate a directional component to the locomotion. In Table IA there is strong evidence for a directional component (the disparity between the experimental and the calculated figures for each test chamber); e.g., for the test at upper right, calculated migration was 58 pm, but the cells actually moved 82 pm. In Table IB, the figures are close to each other, and the locomotion observed is consistent with random locomotion. This simple calculation has been widely, and unjustifiably, ignored by people reporting results of checkerboard filter assays.
[4] A g a r o s e M e t h o d
for Human
Neutrophil
Chemotaxis
B y ROBERT D. NELSON and MICHAEL J. H~RRON
Introduction Chemotaxis of leukocytes under agarose was first described for guinea pig peritoneal neutrophils by Cutler in 1974.1 Soon after, Nelson et al., 2 Johns and Sieber, 3 and Repo 4 described measurement of human neutrophil chemotaxis under agarose. Over the following decade the agarose method, in its original or modified forms, has been used by numerous investigators for measurement of random and/or directed movement of leukocytes as well as cells of other types. In the basic form of the assay, three wells are cut in line in a gel in a tissue culture dish, and cells are placed in the central well with chemoattractant and control medium on opposite sides. Cells migrate radially from the central well by crawling in the space between the agarose and the surface of the dish. After a period of incubation, a normal distribution pattern of migrating cells will be egg shaped with the pointed end of the pattern facing the well containing chemoattractant.
t j. E. Cutler, Proc. Soc. Exp. Biol. Med. 147, 471 (1974). 2 R. D. Nelson, P. G. Quie, a n d R. L. S i m m o n s , J. Immunol. 115, 1650 (1975). 3 T. J. J o h n s a n d O. F. Sieber, Jr., Life Sci. 18, 177 (1976). 4 H. Repo, ScandJ. Irnmunol. 6, 203 (1977).
METHODS IN ENZYMOLOGY, VOL. 162
Copyright© 1988by AcademicPress,Inc. All fightsof reproduction in any form reserved.
[4]
AGAROSE METHOD
51
In this chapter we describe in detail the agarose method we use routinely to measure migratory functions of human neutrophils. Many other versions of the agarose method have been described, however to accommodate special needs. Because these have become too numerous to detail in this chapter, references to some of these methods will be provided together with a discussion of considerations which apply to selection of assay conditions. Methods
Preparation of Agarose Gel Agarose gel is prepared by mixing two solutions prepared separately, one consisting of agarose dissolved in water and the other consisting of double-strength HEPES-buffered tissue culture medium supplemented with gelatin. To prepare agarose medium suffient for four tissue dishes: 1. Dissolve 0.25 g agarose in 12.5 ml of distilled water by heating in a boiling water bath for 10- 15 min. Transfer to a 56 ° water bath. 2. Dissolve 0.125 g of gelatin powder (Difco) in 12.5 ml of doublestrength HEPES-buffered minimal essential medium (MEM) 4a by warming in the 56 ° water bath with occasional swirling. This will require approximately 10 min. [To prepare stock double-stength HEPES-buffered MEM, mix 10 ml of 10× MEM, 1.0 ml stock HEPES buffer (1 M HEPES solution in distilled water adjusted to pH 8.1 by addition of 10 M NaOH), and 39 ml distilled water. Sterilized by filtration and store at 4 °.] 3. Add the gelatin-supplemented MEM to the agarose solution, mix by swirling, and pipet 6-ml volumes to each of four 60 X 15 ml tissue culture dishes. The final agarose medium will be 1% with regard to agarose, 0.5% gelatin, and 1 × MEM. The agarose used in our laboratory is Litex agarose, type HSA, obtained from Accurate Chemical and Scientific Corp., Westbury, NY. Both the source and concentration ofagarose are somewhat arbitrary. Other investigators have used agarose from a variety of sources (i.e., Seakem ME agarose from Marine Colloids, Rockland, ME, and agarose of unspecified types of Calbiochem, San Diego, CA, and Sigma Chemical Co., St. Louis, MO) with apparently compatible success. A choice of agarose may be
4a Abbreviations: MEM, minimal essential medium; AAS, agarose-activated human serum; ZAS, zymosan-activated human serum; fMLP, N-formylmethionylleucylphenylalanine.
52
CHEMOTAXIS
[4]
made in special cases, however, based on a need to eliminate protein 5 or to control the deformability required for cells to enter and migrate in the subagarose space. 2'5 A number of choices are also possible for the tissue culture medium. In addition to MEM, other investigators have used RPMI 1640, Medium 199, or Krebs-Ringer phosphate buffer. No information is available to direct one's choice of medium. The buffer may be either HEPES or bicarbonate, depending on the availability of an incubator with a CO2 atmosphere and the need for a protein-free system2 Although protein may not be an absolute requirement for all agarosebuffer systems, 5 protein supplementation of the gel medium has several advantages. It can provide for easy removal of the gel after fixation and appears to provide for improved cell motility. It also optimizes formation of gradients of purified C5-derived chemoattractants by eliminating nonspecific adsorption of chemoattractant to the substratum. 6 Complementsufficient human serum is not an acceptable protein source for studies of human leukocyte migratory functions because of the ability of agarose to activate the alternate complement pathway. Products of activation of the fifth component of complement in the gel will stimulate random motility and inhibit chemotaxis to C5-derived chemoattractants. Even heat-decomplemented serum is not recommended for this purpose because it will also contain products of complement activation. Any other protein used for this purpose (i.e., ovalbumin, human or bovine serum albumin, or fetal calf serum) should be tested for such influences before its use is accepted. We have not specified a particular kind of tissue culture dish. This is because we have found that dishes from various sources, either treated or untreated for tissue culture, seem to work equally well for assays of human neutrophil migratory functions. For cells of other types, however, this may be an important consideration. The use of glass microscope slides has also been described, 6 providing the advantages of easier viewing and storage. Disadvantages of slides include the requirements for cleaning, pretreatment with gelatin when using certain chemoattractants, 6 and the need to provide a humid atmosphere during setup and incubation.
Cutting Wells Cutting the wells is best done with the aid of a punch and template. Our punch is round with an o.d. of 3.1 mm (11-gauge hypodermic stock) and an inside bevel. Wells of this size easily accept a 10-/d volume of fluid. Our 5 K. W. Mollison, G. W. Carter, and R. A. Krause, Proc. Soc. Exp. Biol. Med. 167, 419 (1981). 6 D. E. Chenoweth, J. G. Rowe, and T. E. Hugli, J. Immunol. Methods 25, 337 (1979).
[4]
AGAROSE METHOD
•
0 0
•
• • 0
• 0 0 •
0 0
•
53
O •
00000 •
• 0
"A" Well: Chemoattractant "B" Well: Cells "C" Well: Control Medium
FI(;. l. Two patterns for cutting wells in agarose gels in tissue culture dishes. Chemoattractant, cells, and control medium are placed in the wells according to the legend. When tissue culture dishes are prepared with 6 ml of agarose gel, wells of 3. l-ram diameter will accept a 10-/zl volume. The C well containing control medium is optional. We recommend that measurements of random motility be derived from either single wells or wells cut in pairs to accept cells and control medium (see text).
template provides for separation of wells in each group by a distance, edge-to-edge, approximating the well diameter. A punch and template can be purchased from the authors, if not otherwise available. Care must be taken not to scratch the surface of the tissue culture dish by twisting the punch or by applying too much pressure. Scratches will provide a barrier for migrating cells. Cores can be extracted using either a hypodermic needle (with the bevel bent to form a hook) or a pipet (with a diameter smaller than the core) attached to a vacuum. Care must also be taken not to lift the surrounding gel from the substrate. Lifting the gel changes the gel-dish interface, allowing easier movement of cells into such areas. To aid in removal of the cores, the dishes can be refrigerated for a short time. When wells are cut and cores removed in advance, fluid will bleed into the prepared wells. This fluid can be removed using either a pipet or a twisted strip of tissue paper as a wick. In some situations, it may be appropriate to increase or decrease the well diameters or the distance between wells. In other situations, rectangular wells or troughs may be preferred to the round wells. 7 Alternative well configurations are diagrammed in Fig. 1. Using threewell arrangements and placing chemoattractant in the well designated A, cells in well B, and control medium in well C provides for measurement of both random and chemotactic migratory functions from the same cell population. Alternative placement of the well containing chemoattractant at the center of the dish will reduce the number of wells needed for the
7 D. A. Lauffenburger, C. Rothman, and S. H. Zigmond, J. lmmunol. 131, 940 (1983).
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CHEMOTAXIS
[4]
chemoattractant. Two-well arrangements of wells A and B can also be used when the control medium does not differ from the medium in the agarose gel and when it may be more appropriate to measure independently the random migratory function of the cells, i.e., when the incubation time and/or chemoattractant concentration allow the gradient to reach the well containing cells.
Applying Cells and Chemoattractants The number of cells added to well B influences their rate of migration and determines the density of the migration pattern. We use 5 × 105 human neutrophils in 10/d, requiring a stock concentration of 5 × 10 7 ceUs/ml. A reduced density of the migration pattern would be an advantage when migratory functions are quantified by enumeration methods 8 or when cellular morphology and orientation are evaluated.9 Chemoattractants used routinely include agarose- or zymosan-activated human serum (AAS or ZAS) as a source of chemotactic product of the fifth component of complement, C5adesArg, and N-formylmethionylleucylphenylalanine (fMLP). Using AAS requires only that complementsufficient serum be added to the appropriate wells and the dishes incubated for 1 hr at 37 ° before addition of the cells. We use undiluted serum for this purpose and suggest that sera from different individuals may vary in terms of chemotactic potential. Increased chemotactic activity of serum may also be achieved by preventing conversion of C5a to C5adesArg through addition of a serum carboxypeptidase inhibitor. DL-2-mercaptomethyl-3-guanidoethylthiopropanoic acid (Calbiochem), 1° or the chemoattractant can be concentrated in the form of ZAS-HCI. ~ We use fMLP dissolved in at a concentration of 1.1 × 10-7 M (5 × 10-5 mg/ml). The arachidonic acid metabolite, leukotriene B4 (LTB4), 12 and cell-free culture supernatants from a variety of microbial species have also been used as chemoattracrants. For any chemoattractant, the concentration used will of necessity be greater than that required to produce a chemotactic response across a membrane filter. This difference is related to the greater distance over which the gradient must be established and the small volume of chemoattractant used with the agarose method.
8 W. Orr and P. A. Ward, J. Immunol. Methods 20, 95 (1978). 9 j. Palmblad, A.-M. Uden, and N. Venizelos, J. Imrnunol. Methods 44, 37 (1981). ~oT. H. Plummer, Jr. and T. J. Ryan, Biochem. Biophys. Res. Commun. 98, 448 (1981). ~1C. Gerard and T. E. Hugli, Biol. Chem. 254, 6346 (1979). tz j. Palmblad, C. L. Malmsten, A.-M. Uden, O. Radmark, L. Engstedt, and B. Samuelsson, Blood 58, 658 (1981).
[4]
AGAROSE METHOD
55
Incubation and Termination The incubation period for routine assays of human neutrophil migratory functions is 2 hr at 37 ° in a humidified atmosphere. For special applications this period has been extended to as long as 8 days? 3 Longer incubation periods demand use of sterile technique and incorporation of antibiotics in the agarose medium. The incubation period will be determined in part by the time over which an effective reagent gradient can be maintained ~3,14and the time over which cells remain viable and retain their motility. At any time during the migration period it is possible to monitor cellular migration microscopically. This can be done by examining the inverted plate using an ordinary microscope, focusing initially on the bottom edge of a well. We advise that this be done routinely before addition of fixative to confirm that cellular migration has occurred. We accomplish termination and fixation by flooding the dishes with 2.5% glutaraldehyde. The fixative is prepared by dilution of a stock 25% aqueous solution (Sigma) in Dulbecco's phosphate-buffered saline. Migration patterns are not preserved if the agarose is removed without prior fixation. Use of formaldehyde, as buffered formalin, and methanol for this purpose is also acceptable but does not provide for optimal preservation of cellular morphology, if such detail is needed. Treatment for either 1 hr at ambient temperature or overnight at 4 ° will fix the cells adequately to the dish to allow removal of the agarose. Removal of the agarose is best done in a hood or ventilated space, holding the dish facing downward at a 45 ° angle, and catching an edge of the gel with a narrow spatula. Do not allow the gel to rotate in the dish during this step or the migration patterns may be smeared. Following removal of the agarose, the dishes are rinsed in tap water and stained with Wright stain or air dried.
Migration Patterns Migration patterns illustrating chemotactic migration patterns of human neutrophils are presented in Fig. 2. Figure 2A represents a typical migration pattern obtained with either AAS or fMLP as the chemoattractant. Figure 2B represents a migration pattern reflecting the blunting which can occur when the concentration of chemoattractant is too high. Grossly high concentrations of a chemoattractant can inhibit directed cellular motility to abrogate a differential response.
~3S. U. Orredson, D. R. Knighton, H. Scheuenstuhl, and T. K. Hunt, J: Surg. Res. 35, 249 (1983). t4 D. A. Lauffenburger and S. H. Zigmond, J. Immunol. Methods 40, 45 (1981).
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CHEMOTAXIS
A
[4]
B
FIG. 2. Two patterns of migration of human neutrophils. (A) Egg-shaped migration pattern typical of a chemotactic response to either AAS or fMLP. The leading edge of the migration pattern toward the source of the chemoattractant may be more or less "rocket shaped," depending in part on the chemoattractant. (B) Blunted migration pattern observed when the concentration of chemoattractant applied is too high. Grossly high concentrations of chemoattractant can produce a migration pattern with a constant radius, falsely indicative of defective chemotaxis.
Quantification of Migratory Functions Quantification of migratory functions can be accomplished by either of two general methods, one involving counting of migrated cells and another involving measurement of the distance of migration, or leading front. Each method has its counterpart in the membrane filter assay. For two methods of measurement involving counting of cells the reader is referred to the report by Orr and Ward? For measurement of migration distance we project migration patterns onto a piece of graph paper using a Tri-Simplex microprojector (Bausch and Lomb) fitted with a 12 X objective, giving a final magnification of approximately X 40. This information can alternatively be obtained using a microscope fitted with an appropriate ocular micrometer disk? In either
[4]
AGAROSE METHOD
57
+ VUAW~
lIB!!
FIG. 3. Quantification of migratory functions by the leading-front method. The distance of cellular migration from the edge of the central well outward toward the well containing chemoattractant, distance A, represents chemotaxis. The distance of cellular migration from the edge of the well outward toward the well containing control medium, distance B, represents random motility. These distances can be measured macroscopicaUy with the aid of a projecting microscope or microscopically using a microscope with grid disk inserted in the ocular lens. The text describes methods of manipulation of these distance measurements to provide a chemotactic differential or chemotactie index.
case, the distance of cellular migration outward from the well margin is recorded as illustrated in Fig. 3. The distal limit of the migration pattern can be identified subjectively by ignoring cells that have "beaten the pack." It can be identified more objectively by excluding some specified number of front-runner cells 5 or by defining the limit as the farthest point at which some number of cells (i.e., three) are aligned in the same plan and parallel to the margin of the central well3 For measurements of chemotactic responses, the migration pattern should be positioned on the graph paper so that the measurement is taken on the axis joining the well centers. Our projected (×40) and actual (× 1) values for random and chemotactic migration distances fall within the ranges described in Table I. TABLE I
RANDOM AND CHEMOTACTIC MIGRATION DISTANCES Distance Function
Well
X40 (cm)
× 1 (mm)
Random motility Chemotaxis, ZAS Chemotaxis, fMLP
B A A
2.5 to 4.5 3.5 to 5.5 6.5 to 9.0
0.6 to 1.1 0.9 to 1.4 1.6 to 2.3
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CHEMOTAXIS
[4]
In some situations further manipulation of migration distance data may be attempted. For example, patient neutrophils or neutrophils receiving in vitro pretreatments often exhibit reduced distances of migration for all migratory functions tested. In this situation differentiation between loss of chemotactic function related nonspecifically to loss of random motility or specifically to loss of responsiveness to an individual chemoattractant is not possible by consideration of the separate migration distance values. However, by calculation of a "chemotactic differential" or a "chemotactic index," such differentiation may be attempted. These calculations are illustrated below. Chemotactic differential = distance chemotaxis distance random motility distance chemotaxis Chemotactic index (alternative 1) - distance random motility -
distance chemotaxis distance random motility Chemotactic index (alternative 2) distance random motility -
Of these, we prefer chemotactic index (alternative 2) for this purpose because the chemotaxis value is corrected for its random motility component before the two functions are related.
Troubleshooting There will be sporadic occasions when cell migration will not occur and no explanation can be found for the result. This is not atypical of bioassays. Repeated failure to observe cell migration, however, seems to occur for one of two identifiable reasons. One is improper preparation of the tissue culture medium to be mixed with the agarose. This must be doublestrength medium so that dilution with the agarose yields an isotonic solution. The other involves handling of the neutrophils and seems most often to involve either the isolation protocol or hypotonic lysis of erythrocytes. To test these possibilities, we recommend use of neutrophils as buffy coat leukocytes or purified neutrophils without elimination of erythrocytes.
Special Applications The agarose method provides the investigator with opportunities for analysis of cellular migration which are either not available or at best difficult with the membrane filter assay. These include opportunities for examining migratory functions by microcinematography and cellular fea-
[5]
51Cr CHEMOTAXIS ASSAY
59
tures by both scanning ~5 and transmission electron microscopy (unpublished observations). To facilitate sectioning for TEM analysis, cells are allowed to migrate on a Thermanox coverslip (Miles Scientific). The agarose method can also be used for analyses of the random and directional component of leukocyte chemotaxis. Two reports describe measurement of cell orientation toward the chemoattractant. Palmblad et al. 9 have applied the visual chemotaxis assay of Zigmond and Hirsch ~6 to obtain this information, and MacFarlane et al. 17 have applied computerinterfaced video microscopy to analysis of cell shape and orientation. The latter authors descibe a "chemotactic behavior index" which compares abnormal with control neutrophil chemotactic function using both cell orientation and migration distance data together. Reports by Lauffenburger and colleagues7,~8 describe a more sophisticated mathematical approach to analysis of the chemosensory movement of cells. A formula incorporating random motility and chemotaxis coefficients together with attractant concentration provides for precise quantitative characterization of the relative contributions of chemokinesis and chemotaxis to population migration as well as for future investigation of the consequences of changes in speed, persistence time, and orientation bias on this phenomenon. Space does not allow for a review of conditions which have been applied to studies of migratory functions of cells of other types from both human and other sources. For information on such applications of the agarose method, the reader is invited to contact the authors. 15 R. D. Nelson, V. D. Fiegel, and R. L. Simmons, J. Immunol. 117, 1676 (1976). 16 S. H. Zigmond and J. G. Hirsch, J. Exp. Med. 137, 387 (1973). 17 G. D. MacFarlane, M. C. Herzberg, and R. D. Nelson, J. Leukocyte Biol. 41, 307 (1987). 18 R. T. Tranquillo, S. H. Zigmond, and D. A. Lauffenburger, "Cell Motility and the Cytoskele,ton." In Press, 1988.
[5] C h r o m i u m - 5 1
Radioimmunoassay
for Chemotaxis
B y JOHN I. GALLIN
Introduction The SlCr radioassay for chemotaxis was introduced over 15 years ago for the study of neutrophil locomotion.l-3 Subsequently, it has been modii E. J. Goetzl and K. F. Austen, Immunol. Commun. 1, 421 (1972). 2 j. I. Gallin, R. A. Clark, and H. R. Kimball, J. Immunol. 110, 233 (1973), 3 j. I. Gallin, Antibiot. Chemother. (Washington, D.C.) 19, 146 (1974).
METHODS IN ENZYMOLOGY, VOL. 162
Copyright © 1988 by Academic Press, lnc, All rights of reproduction in any form reserved,
CHEMOTAXIS
[4]
This is the figure we would expect on the basis of random migration alone, and the figures in parentheses in Table I are based on similar calculations (but with more points, i.e., Vo, V1, V2, 1/3, etc. and dl, dE, d3, etc. (based on the figures on the diagonals). If, in an actual experiment, the cells had moved 95/zm/hr instead of 68/tm in a linear gradient from 0 to 10/zg/ml, this would be much higher than would be expected from random locomotion alone, and would indicate a directional component to the locomotion. In Table IA there is strong evidence for a directional component (the disparity between the experimental and the calculated figures for each test chamber); e.g., for the test at upper right, calculated migration was 58 pm, but the cells actually moved 82 pm. In Table IB, the figures are close to each other, and the locomotion observed is consistent with random locomotion. This simple calculation has been widely, and unjustifiably, ignored by people reporting results of checkerboard filter assays.
[4] A g a r o s e M e t h o d
for Human
Neutrophil
Chemotaxis
B y ROBERT D. NELSON and MICHAEL J. H~RRON
Introduction Chemotaxis of leukocytes under agarose was first described for guinea pig peritoneal neutrophils by Cutler in 1974.1 Soon after, Nelson et al., 2 Johns and Sieber, 3 and Repo 4 described measurement of human neutrophil chemotaxis under agarose. Over the following decade the agarose method, in its original or modified forms, has been used by numerous investigators for measurement of random and/or directed movement of leukocytes as well as cells of other types. In the basic form of the assay, three wells are cut in line in a gel in a tissue culture dish, and cells are placed in the central well with chemoattractant and control medium on opposite sides. Cells migrate radially from the central well by crawling in the space between the agarose and the surface of the dish. After a period of incubation, a normal distribution pattern of migrating cells will be egg shaped with the pointed end of the pattern facing the well containing chemoattractant.
t j. E. Cutler, Proc. Soc. Exp. Biol. Med. 147, 471 (1974). 2 R. D. Nelson, P. G. Quie, a n d R. L. S i m m o n s , J. Immunol. 115, 1650 (1975). 3 T. J. J o h n s a n d O. F. Sieber, Jr., Life Sci. 18, 177 (1976). 4 H. Repo, ScandJ. Irnmunol. 6, 203 (1977).
METHODS IN ENZYMOLOGY, VOL. 162
Copyright© 1988by AcademicPress,Inc. All fightsof reproduction in any form reserved.
[4]
AGAROSE METHOD
51
In this chapter we describe in detail the agarose method we use routinely to measure migratory functions of human neutrophils. Many other versions of the agarose method have been described, however to accommodate special needs. Because these have become too numerous to detail in this chapter, references to some of these methods will be provided together with a discussion of considerations which apply to selection of assay conditions. Methods
Preparation of Agarose Gel Agarose gel is prepared by mixing two solutions prepared separately, one consisting of agarose dissolved in water and the other consisting of double-strength HEPES-buffered tissue culture medium supplemented with gelatin. To prepare agarose medium suffient for four tissue dishes: 1. Dissolve 0.25 g agarose in 12.5 ml of distilled water by heating in a boiling water bath for 10- 15 min. Transfer to a 56 ° water bath. 2. Dissolve 0.125 g of gelatin powder (Difco) in 12.5 ml of doublestrength HEPES-buffered minimal essential medium (MEM) 4a by warming in the 56 ° water bath with occasional swirling. This will require approximately 10 min. [To prepare stock double-stength HEPES-buffered MEM, mix 10 ml of 10× MEM, 1.0 ml stock HEPES buffer (1 M HEPES solution in distilled water adjusted to pH 8.1 by addition of 10 M NaOH), and 39 ml distilled water. Sterilized by filtration and store at 4 °.] 3. Add the gelatin-supplemented MEM to the agarose solution, mix by swirling, and pipet 6-ml volumes to each of four 60 X 15 ml tissue culture dishes. The final agarose medium will be 1% with regard to agarose, 0.5% gelatin, and 1 × MEM. The agarose used in our laboratory is Litex agarose, type HSA, obtained from Accurate Chemical and Scientific Corp., Westbury, NY. Both the source and concentration ofagarose are somewhat arbitrary. Other investigators have used agarose from a variety of sources (i.e., Seakem ME agarose from Marine Colloids, Rockland, ME, and agarose of unspecified types of Calbiochem, San Diego, CA, and Sigma Chemical Co., St. Louis, MO) with apparently compatible success. A choice of agarose may be
4a Abbreviations: MEM, minimal essential medium; AAS, agarose-activated human serum; ZAS, zymosan-activated human serum; fMLP, N-formylmethionylleucylphenylalanine.
52
CHEMOTAXIS
[4]
made in special cases, however, based on a need to eliminate protein 5 or to control the deformability required for cells to enter and migrate in the subagarose space. 2'5 A number of choices are also possible for the tissue culture medium. In addition to MEM, other investigators have used RPMI 1640, Medium 199, or Krebs-Ringer phosphate buffer. No information is available to direct one's choice of medium. The buffer may be either HEPES or bicarbonate, depending on the availability of an incubator with a CO2 atmosphere and the need for a protein-free system2 Although protein may not be an absolute requirement for all agarosebuffer systems, 5 protein supplementation of the gel medium has several advantages. It can provide for easy removal of the gel after fixation and appears to provide for improved cell motility. It also optimizes formation of gradients of purified C5-derived chemoattractants by eliminating nonspecific adsorption of chemoattractant to the substratum. 6 Complementsufficient human serum is not an acceptable protein source for studies of human leukocyte migratory functions because of the ability of agarose to activate the alternate complement pathway. Products of activation of the fifth component of complement in the gel will stimulate random motility and inhibit chemotaxis to C5-derived chemoattractants. Even heat-decomplemented serum is not recommended for this purpose because it will also contain products of complement activation. Any other protein used for this purpose (i.e., ovalbumin, human or bovine serum albumin, or fetal calf serum) should be tested for such influences before its use is accepted. We have not specified a particular kind of tissue culture dish. This is because we have found that dishes from various sources, either treated or untreated for tissue culture, seem to work equally well for assays of human neutrophil migratory functions. For cells of other types, however, this may be an important consideration. The use of glass microscope slides has also been described, 6 providing the advantages of easier viewing and storage. Disadvantages of slides include the requirements for cleaning, pretreatment with gelatin when using certain chemoattractants, 6 and the need to provide a humid atmosphere during setup and incubation.
Cutting Wells Cutting the wells is best done with the aid of a punch and template. Our punch is round with an o.d. of 3.1 mm (11-gauge hypodermic stock) and an inside bevel. Wells of this size easily accept a 10-/d volume of fluid. Our 5 K. W. Mollison, G. W. Carter, and R. A. Krause, Proc. Soc. Exp. Biol. Med. 167, 419 (1981). 6 D. E. Chenoweth, J. G. Rowe, and T. E. Hugli, J. Immunol. Methods 25, 337 (1979).
[4]
AGAROSE METHOD
•
0 0
•
• • 0
• 0 0 •
0 0
•
53
O •
00000 •
• 0
"A" Well: Chemoattractant "B" Well: Cells "C" Well: Control Medium
FI(;. l. Two patterns for cutting wells in agarose gels in tissue culture dishes. Chemoattractant, cells, and control medium are placed in the wells according to the legend. When tissue culture dishes are prepared with 6 ml of agarose gel, wells of 3. l-ram diameter will accept a 10-/zl volume. The C well containing control medium is optional. We recommend that measurements of random motility be derived from either single wells or wells cut in pairs to accept cells and control medium (see text).
template provides for separation of wells in each group by a distance, edge-to-edge, approximating the well diameter. A punch and template can be purchased from the authors, if not otherwise available. Care must be taken not to scratch the surface of the tissue culture dish by twisting the punch or by applying too much pressure. Scratches will provide a barrier for migrating cells. Cores can be extracted using either a hypodermic needle (with the bevel bent to form a hook) or a pipet (with a diameter smaller than the core) attached to a vacuum. Care must also be taken not to lift the surrounding gel from the substrate. Lifting the gel changes the gel-dish interface, allowing easier movement of cells into such areas. To aid in removal of the cores, the dishes can be refrigerated for a short time. When wells are cut and cores removed in advance, fluid will bleed into the prepared wells. This fluid can be removed using either a pipet or a twisted strip of tissue paper as a wick. In some situations, it may be appropriate to increase or decrease the well diameters or the distance between wells. In other situations, rectangular wells or troughs may be preferred to the round wells. 7 Alternative well configurations are diagrammed in Fig. 1. Using threewell arrangements and placing chemoattractant in the well designated A, cells in well B, and control medium in well C provides for measurement of both random and chemotactic migratory functions from the same cell population. Alternative placement of the well containing chemoattractant at the center of the dish will reduce the number of wells needed for the
7 D. A. Lauffenburger, C. Rothman, and S. H. Zigmond, J. lmmunol. 131, 940 (1983).
54
CHEMOTAXIS
[4]
chemoattractant. Two-well arrangements of wells A and B can also be used when the control medium does not differ from the medium in the agarose gel and when it may be more appropriate to measure independently the random migratory function of the cells, i.e., when the incubation time and/or chemoattractant concentration allow the gradient to reach the well containing cells.
Applying Cells and Chemoattractants The number of cells added to well B influences their rate of migration and determines the density of the migration pattern. We use 5 × 105 human neutrophils in 10/d, requiring a stock concentration of 5 × 10 7 ceUs/ml. A reduced density of the migration pattern would be an advantage when migratory functions are quantified by enumeration methods 8 or when cellular morphology and orientation are evaluated.9 Chemoattractants used routinely include agarose- or zymosan-activated human serum (AAS or ZAS) as a source of chemotactic product of the fifth component of complement, C5adesArg, and N-formylmethionylleucylphenylalanine (fMLP). Using AAS requires only that complementsufficient serum be added to the appropriate wells and the dishes incubated for 1 hr at 37 ° before addition of the cells. We use undiluted serum for this purpose and suggest that sera from different individuals may vary in terms of chemotactic potential. Increased chemotactic activity of serum may also be achieved by preventing conversion of C5a to C5adesArg through addition of a serum carboxypeptidase inhibitor. DL-2-mercaptomethyl-3-guanidoethylthiopropanoic acid (Calbiochem), 1° or the chemoattractant can be concentrated in the form of ZAS-HCI. ~ We use fMLP dissolved in at a concentration of 1.1 × 10-7 M (5 × 10-5 mg/ml). The arachidonic acid metabolite, leukotriene B4 (LTB4), 12 and cell-free culture supernatants from a variety of microbial species have also been used as chemoattracrants. For any chemoattractant, the concentration used will of necessity be greater than that required to produce a chemotactic response across a membrane filter. This difference is related to the greater distance over which the gradient must be established and the small volume of chemoattractant used with the agarose method.
8 W. Orr and P. A. Ward, J. Immunol. Methods 20, 95 (1978). 9 j. Palmblad, A.-M. Uden, and N. Venizelos, J. Imrnunol. Methods 44, 37 (1981). ~oT. H. Plummer, Jr. and T. J. Ryan, Biochem. Biophys. Res. Commun. 98, 448 (1981). ~1C. Gerard and T. E. Hugli, Biol. Chem. 254, 6346 (1979). tz j. Palmblad, C. L. Malmsten, A.-M. Uden, O. Radmark, L. Engstedt, and B. Samuelsson, Blood 58, 658 (1981).
[4]
AGAROSE METHOD
55
Incubation and Termination The incubation period for routine assays of human neutrophil migratory functions is 2 hr at 37 ° in a humidified atmosphere. For special applications this period has been extended to as long as 8 days? 3 Longer incubation periods demand use of sterile technique and incorporation of antibiotics in the agarose medium. The incubation period will be determined in part by the time over which an effective reagent gradient can be maintained ~3,14and the time over which cells remain viable and retain their motility. At any time during the migration period it is possible to monitor cellular migration microscopically. This can be done by examining the inverted plate using an ordinary microscope, focusing initially on the bottom edge of a well. We advise that this be done routinely before addition of fixative to confirm that cellular migration has occurred. We accomplish termination and fixation by flooding the dishes with 2.5% glutaraldehyde. The fixative is prepared by dilution of a stock 25% aqueous solution (Sigma) in Dulbecco's phosphate-buffered saline. Migration patterns are not preserved if the agarose is removed without prior fixation. Use of formaldehyde, as buffered formalin, and methanol for this purpose is also acceptable but does not provide for optimal preservation of cellular morphology, if such detail is needed. Treatment for either 1 hr at ambient temperature or overnight at 4 ° will fix the cells adequately to the dish to allow removal of the agarose. Removal of the agarose is best done in a hood or ventilated space, holding the dish facing downward at a 45 ° angle, and catching an edge of the gel with a narrow spatula. Do not allow the gel to rotate in the dish during this step or the migration patterns may be smeared. Following removal of the agarose, the dishes are rinsed in tap water and stained with Wright stain or air dried.
Migration Patterns Migration patterns illustrating chemotactic migration patterns of human neutrophils are presented in Fig. 2. Figure 2A represents a typical migration pattern obtained with either AAS or fMLP as the chemoattractant. Figure 2B represents a migration pattern reflecting the blunting which can occur when the concentration of chemoattractant is too high. Grossly high concentrations of a chemoattractant can inhibit directed cellular motility to abrogate a differential response.
~3S. U. Orredson, D. R. Knighton, H. Scheuenstuhl, and T. K. Hunt, J: Surg. Res. 35, 249 (1983). t4 D. A. Lauffenburger and S. H. Zigmond, J. Immunol. Methods 40, 45 (1981).
56
CHEMOTAXIS
A
[4]
B
FIG. 2. Two patterns of migration of human neutrophils. (A) Egg-shaped migration pattern typical of a chemotactic response to either AAS or fMLP. The leading edge of the migration pattern toward the source of the chemoattractant may be more or less "rocket shaped," depending in part on the chemoattractant. (B) Blunted migration pattern observed when the concentration of chemoattractant applied is too high. Grossly high concentrations of chemoattractant can produce a migration pattern with a constant radius, falsely indicative of defective chemotaxis.
Quantification of Migratory Functions Quantification of migratory functions can be accomplished by either of two general methods, one involving counting of migrated cells and another involving measurement of the distance of migration, or leading front. Each method has its counterpart in the membrane filter assay. For two methods of measurement involving counting of cells the reader is referred to the report by Orr and Ward? For measurement of migration distance we project migration patterns onto a piece of graph paper using a Tri-Simplex microprojector (Bausch and Lomb) fitted with a 12 X objective, giving a final magnification of approximately X 40. This information can alternatively be obtained using a microscope fitted with an appropriate ocular micrometer disk? In either
[4]
AGAROSE METHOD
57
+ VUAW~
lIB!!
FIG. 3. Quantification of migratory functions by the leading-front method. The distance of cellular migration from the edge of the central well outward toward the well containing chemoattractant, distance A, represents chemotaxis. The distance of cellular migration from the edge of the well outward toward the well containing control medium, distance B, represents random motility. These distances can be measured macroscopicaUy with the aid of a projecting microscope or microscopically using a microscope with grid disk inserted in the ocular lens. The text describes methods of manipulation of these distance measurements to provide a chemotactic differential or chemotactie index.
case, the distance of cellular migration outward from the well margin is recorded as illustrated in Fig. 3. The distal limit of the migration pattern can be identified subjectively by ignoring cells that have "beaten the pack." It can be identified more objectively by excluding some specified number of front-runner cells 5 or by defining the limit as the farthest point at which some number of cells (i.e., three) are aligned in the same plan and parallel to the margin of the central well3 For measurements of chemotactic responses, the migration pattern should be positioned on the graph paper so that the measurement is taken on the axis joining the well centers. Our projected (×40) and actual (× 1) values for random and chemotactic migration distances fall within the ranges described in Table I. TABLE I
RANDOM AND CHEMOTACTIC MIGRATION DISTANCES Distance Function
Well
X40 (cm)
× 1 (mm)
Random motility Chemotaxis, ZAS Chemotaxis, fMLP
B A A
2.5 to 4.5 3.5 to 5.5 6.5 to 9.0
0.6 to 1.1 0.9 to 1.4 1.6 to 2.3
58
CHEMOTAXIS
[4]
In some situations further manipulation of migration distance data may be attempted. For example, patient neutrophils or neutrophils receiving in vitro pretreatments often exhibit reduced distances of migration for all migratory functions tested. In this situation differentiation between loss of chemotactic function related nonspecifically to loss of random motility or specifically to loss of responsiveness to an individual chemoattractant is not possible by consideration of the separate migration distance values. However, by calculation of a "chemotactic differential" or a "chemotactic index," such differentiation may be attempted. These calculations are illustrated below. Chemotactic differential = distance chemotaxis distance random motility distance chemotaxis Chemotactic index (alternative 1) - distance random motility -
distance chemotaxis distance random motility Chemotactic index (alternative 2) distance random motility -
Of these, we prefer chemotactic index (alternative 2) for this purpose because the chemotaxis value is corrected for its random motility component before the two functions are related.
Troubleshooting There will be sporadic occasions when cell migration will not occur and no explanation can be found for the result. This is not atypical of bioassays. Repeated failure to observe cell migration, however, seems to occur for one of two identifiable reasons. One is improper preparation of the tissue culture medium to be mixed with the agarose. This must be doublestrength medium so that dilution with the agarose yields an isotonic solution. The other involves handling of the neutrophils and seems most often to involve either the isolation protocol or hypotonic lysis of erythrocytes. To test these possibilities, we recommend use of neutrophils as buffy coat leukocytes or purified neutrophils without elimination of erythrocytes.
Special Applications The agarose method provides the investigator with opportunities for analysis of cellular migration which are either not available or at best difficult with the membrane filter assay. These include opportunities for examining migratory functions by microcinematography and cellular fea-
[5]
51Cr CHEMOTAXIS ASSAY
59
tures by both scanning ~5 and transmission electron microscopy (unpublished observations). To facilitate sectioning for TEM analysis, cells are allowed to migrate on a Thermanox coverslip (Miles Scientific). The agarose method can also be used for analyses of the random and directional component of leukocyte chemotaxis. Two reports describe measurement of cell orientation toward the chemoattractant. Palmblad et al. 9 have applied the visual chemotaxis assay of Zigmond and Hirsch ~6 to obtain this information, and MacFarlane et al. 17 have applied computerinterfaced video microscopy to analysis of cell shape and orientation. The latter authors descibe a "chemotactic behavior index" which compares abnormal with control neutrophil chemotactic function using both cell orientation and migration distance data together. Reports by Lauffenburger and colleagues7,~8 describe a more sophisticated mathematical approach to analysis of the chemosensory movement of cells. A formula incorporating random motility and chemotaxis coefficients together with attractant concentration provides for precise quantitative characterization of the relative contributions of chemokinesis and chemotaxis to population migration as well as for future investigation of the consequences of changes in speed, persistence time, and orientation bias on this phenomenon. Space does not allow for a review of conditions which have been applied to studies of migratory functions of cells of other types from both human and other sources. For information on such applications of the agarose method, the reader is invited to contact the authors. 15 R. D. Nelson, V. D. Fiegel, and R. L. Simmons, J. Immunol. 117, 1676 (1976). 16 S. H. Zigmond and J. G. Hirsch, J. Exp. Med. 137, 387 (1973). 17 G. D. MacFarlane, M. C. Herzberg, and R. D. Nelson, J. Leukocyte Biol. 41, 307 (1987). 18 R. T. Tranquillo, S. H. Zigmond, and D. A. Lauffenburger, "Cell Motility and the Cytoskele,ton." In Press, 1988.
[5] C h r o m i u m - 5 1
Radioimmunoassay
for Chemotaxis
B y JOHN I. GALLIN
Introduction The SlCr radioassay for chemotaxis was introduced over 15 years ago for the study of neutrophil locomotion.l-3 Subsequently, it has been modii E. J. Goetzl and K. F. Austen, Immunol. Commun. 1, 421 (1972). 2 j. I. Gallin, R. A. Clark, and H. R. Kimball, J. Immunol. 110, 233 (1973), 3 j. I. Gallin, Antibiot. Chemother. (Washington, D.C.) 19, 146 (1974).
METHODS IN ENZYMOLOGY, VOL. 162
Copyright © 1988 by Academic Press, lnc, All rights of reproduction in any form reserved,