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 (seeHint #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 10⁷cells 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.