The Under-Agarose Migration Assay

互联网2013-09-05

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overviewThe Under-Agarose assay is a useful method for observing the response of a cell population to one or more chemoattractant sources. The behavior of individual migrating cells can be studied by modifying the assay for video microscopy. The assay works well with freshly-isolated human neutropils. Monocytes can also be observed to migrate but require more time. It is unclear whether lymphocytes can be induced to migrate in this assay. The procedure makes use of a tissue culture dish filled with an agarose mixture. Chemoattractant diffuses from wells in the agarose to form a gradient. Cells in nearby wells can be monitored as they migrate in the direction of the chemoattractant source.

Procedure

A. Preparation of Agarose Filled Plates

1. Prepare HBSS-Agarose Solution and RPMI/BCS Solution, and equilibrate both to 50°C (see Hint #1 ).

2. Prepare Agarose Solution and 2X HEPES Solution, and equilibrate both to 50°C.

3. Mix the two solutions from step #3 or #4, respectively, at a 1:1 ratio.

4. Pour the agarose mixture into 35 x 100 mm tissue culture dishes (3 ml/dish). Larger dishes may be used with volumes adjusted accordingly.

7. Allow the mixture to solidify and place the plates in a humidified 37°C incubator for several hours or overnight.

B. Cutting the Wells

1. Prepare five wells in a straight line, 3mm in diameter and separated by 2.2 mm, with a template.

2. The template is crafted from a piece of metal, containing precisely-positioned holes, that has been cut to fit over the tissue culture dish (see Hint #3 ).

3. To cut the wells, attach a sterile implement that cuts a 3 mm hole, such as a steel punch or a plastic pipette tip, and cut to the proper diameter of the vacuum line (see Hint #4 ).

4. Push the implement through the Agarose layer until it reaches the plastic plate.

5. Cut the wells no more than several hours before the assay to avoid drying the agarose.

C. Performing the Assay

1. Dilute the chemoattractants (see Hint #5 ) and resuspend the cells in migration medium at 10⁷ cells per ml. Use Migration Medium with plates prepared with RPMI/BCS and HEPES Migration Medium with plates prepared with 2X HEPES Migration Medium.

2. Fill the two most peripheral wells with 10 μl of the appropriate Migration Medium.

3. Place 10 μl of the (10⁵ ) cells in two intermediate wells.

4. Place 10 μl of the chemoattractant solution in the central well.

5. Return the plate to the 37° CO₂ incubator for two hours for neutrophils or longer for other cell types (see Hint #6 ).

D. Fixing and Staining

1. After incubation, flood each plate with 1 ml of absolute Methanol. Allow cells to fix for 30 minutes at room temperature or at 4°C overnight.

2. Pour off the Methanol and flood each plate with 1 ml of 37% Formaldehyde. Allow the cells to fix for 30 minutes at room temperature (or longer if at 4°C).

3. Remove the Agarose. The cells should now be fixed to the plastic dish.

4. Stain by adding 1 ml of Fields Stain B (0.5% w/v) per plate.

5. Next, add 1 ml Fields Stain A (2.5% w/v) per plate.

6. Rinse plates and set them aside to dry.

E. Modifications of the Basic Assay

1. The basic assay can be modified to ask more specific questions, such as how neutrophils migrate when presented with two chemoattractant sources instead of one.

F. Video Microscopy

1. The behavior of migrating cells may be filmed with an inverted microscope fitted with a CCD camera.

2. Determine the best magnification, contrast, etc., to observe the cells through the microscope.

3. Keep the plates at 37°C with a microscope stage warmer during the assay.

4. To prevent the plates from drying out, a simple humidified chamber can be made by snugly fitting a larger plastic dish rimmed with wet paper towels over the chemotaxis plate.

5. To keep cells at the proper pH during the assay, use HEPES-buffered Agarose and HEPES-Migration Medium as described.

G. Assessing Results: Do the Cells Migrate Towards the Chemoattractant Source Well?

1. Measuring the leading edge distance is one simple way to describe the effect of the chemoattractant on the cell migration pattern. Measure the distance from the edge of the cell starting well to the leading edge of the migrating cell front.

2. Compare the distance of the cell migration towards the chemoattractant well with the distance of the cell migration towards a well that only contains medium (see Hint #7 ).

3. The number of cells that have been induced to migrate is important for determining the effects of an inhibitor of cell migration, or for comparing the efficacy of one chemoattractant to that of another. To do this, count the large numbers of cells that migrate in the entire assay or, more easily, count the number of cells that have migrated into a particular region of the plate.

4. Measure cell density by assessing the number of cells that have migrated into squares of a counting grid at different distances from the cell starting well (see Hint #8 ).

5. Use a template to count the number of cells that have obtained a specific position with respect to the cell starting well (see Hint #9 ).

H. Assessing Results: How Much of the Observed Migration Is Due to Chemotaxis Versus Chemokinesis?

1. Measure chemoattractant-stimulated motility (chemokinesis) by observing the effect of a uniform field of chemoattractant (i.e., incorporated into the agarose) on cell migration at a variety of different concentrations (see Hint #10 ).

2. These measurements can determine a chemokinesis coefficient, which can be used to calculate the relative contribution of chemokinesis and chemotaxis to the cell migration pattern in a chemoattractant gradient.

3. The orientation and migration of individual cells can be observed under video microscopy.

4. Nuclear orientation can also be employed to assess whether or not fixed cells are oriented towards the chemoattractant source well.

I. Trouble Shooting: When Cells Don't Migrate

1. What chemoattractant concentration should be used? Often, a chemoattractant source of 10 microliters of a solution 10-100 times the EC₅₀ for chemotaxis in a filter assay induces optimal migration in the Under-Agarose assay. Be sure to test a range of chemoattractant doses.

2. When should the cells be isolated? With neutrophils, it is best to begin the assay within 2 to 3 hours of drawing the blood for the neutrophil isolation. Neutrophils should be kept on ice until the start of the assay.

Solutions

Migration Medium		Prepare in RPMI-1640 medium 10% (v/v) BCS, heat inactivated or 0.5% (w/v) BSA

RPMI/BCS Solution		20% (v/v) bovine calf serum (BCS), heat inactivated or 1% (w/v) bovine serum albumin (BSA) Prepare in RPMI-1640 medium

HBSS-Agarose		2.4% (w/v) agarose Prepare by boiling in Hanks' Balanced Salt Solution (HBSS)

2.5% (w/v) Fields Stain A

O.5% (w/v) Fields Stain B

37% Formaldehyde

RPMI-1640 Medium

HEPES-Migration Medium		Prepare in 1X HEPES Solution 10% (v/v) BCS, heat inactivated or 0.5% (w/v) BSA

2X HEPES Solution		20% (v/v) bovine calf serum, heat inactivated or 1% (w/v) bovine serum albumin 20mM HEPES 2X RPMI without bicarbonate (10X available from SIGMA)

Agarose Solution		Prepare by boiling in sterile water 2.4% (w/v) Agarose

BioReagents and Chemicals

Hanks' Balanced Salt Solution (HBSS)
Fields Stain B
Fields Stain A
Bovine Serum Albumin
Methanol
Formaldehyde
RPMI-1640
Agarose
Bovine Calf Serum
HEPES
Chemoattractant
Heat-Inactivated Serum
Protocol Hints

1. Serum is heat-inactivated to destroy labile chemotatic factors, such as complement component C5a. Because serum itself can be chemotactic, some investigators prefer to use serum albumin instead. Both serum and serum albumin can stimulate cell motility. To avoid these effects, gelatin may be used as a substitute for protein.

Plates contain protein (see Hint #1 ), approximately 1% Agarose and balanced salt solution, and/or tissue culture medium. Heat-Inactivated Serum may be used as a protein source.

Plates should be maintained at physiological pH (7.4) during the assay. If plates will not be kept in a CO₂ incubator (i.e., for video microscopy), skip to Step 4 to prepare HEPES buffered plates.

2. If plates will not be kept in a CO₂ incubator (i.e., for video microscopy), 10 mM HEPES that has been titrated to the correct pH can be incorporated into the plates.

3. As illustrated in Nelson et al., (see Citation #2 ), the metal template is placed over the Agarose-containing dish and a steel punch is inserted in the holes to cut the desired pattern of wells. As a simpler, low-tech alternative, an actual-size diagram of the desired well pattern can be placed underneath the dish and used as a guide for well-cutting.

4. If using a steel punch, avoid creating scratches in the plastic, which could become barriers to cell migration.

5. Often, a chemattractant source of 10 microliters of a solution 10-100 times the EC₅₀ for chemotaxis in a filter assay induces optimal migration in the Under-Agarose assay. Be sure to test a range of chemoattractant doses.

6. Migrating cells can be observed during or after the chemotaxis assay through an inverted microscope.

7. A useful tool for performing these measurements is a magnifier with reticles, (available from Fisher Scientific).

8. This method allows comparison between the number of cells migrating in one assay with those of another assay. A plot of cell density at different distances from the starting well provides more information about cell migratory behavior than an estimate of the leading front distance alone.

9. This may be of interest particularly when assessing where cells migrate when they encounter multiple chemoattractant sources.

10. Since many chemoattractants stimulate cell motility in a dose-dependent manner, a careful analysis is required to distinguish chemoattractant-stimulated motility from directional migration or chemotaxis.

2. Nelson, RD, Quie, PG, Simmons, RL. A New and Simple Method for Measuring Chemotaxis and Spontaneous Migration of Human Polymorphonuclear Leukocytes and Monocytes. J. Immunol. 1975; 115: 1650-1656

1. Foxman, EF, Campbell, JJ, and Butcher, EC. Multi-step navigation and the combinatorial control of leukocyte chemotaxis. J. Cell Biol. 1997; 139: 1349-1360.