| Overview
| |
| This assay uses a simple apparatus to study chemotaxis of leukocytes or other migratory cells. The apparatus consists of two multi-well chambers separated by a filter containing pores of uniform size. A solution containing a chemokine or chemotactic factor is placed in the botton chamber and a cell suspension is placed in the upper chamber. Cells can migrate through the pores, across the thickness of the filter, and toward the source of chemoattractant (the lower chamber). Cells that migrated across the filter and attached to the underside are counted. Data is often expressed in terms of Migration Index: the number of cells that migrated in response to agonist relative to the number of cells that migrated randomly, that is, to buffer only. | |
| Procedure
| |
| A. Filter Preparation (see Hint #2) 1. This procedure uses 25 x 80 mm polycarbonate filters, polyvinylpropylene (PVP) free, pore size 3, 5, 8 or 10 μm (Poretics or Neuro Probes) (see Hint #3). If using leukocytes, skip to Section B. 2. Prepare 3 to 5 ml of Rat Tail Collagen Solution. 3. Pour the Rat Tail Collagen Solution into a shallow vessel and submerge the filters in this solution with forceps (see Hint #4 and Hint #5). 4. Incubate for 2 hr at 37°C. 5. Remove the filter from the solution with forceps and allow to dry on a Kimwipe. B. Cell Preparation 1. Grow adherent cells to 80% confluence. For cells grown in suspension, skip to step 6. 2. Wash cells once with 2 to 3 ml per 10 cm dish of Trypsin/EDTA Solution. Aspirate solution immediately. 3. Add 2 ml of Trypsin/EDTA Solution per 10 cm dish. Incubate for 2 minutes at room temperature. 4. Add 1 ml of Cell Dissociation Buffer. Incubate for 2 minutes at room temperature. 5. Add 6 ml of Serum Containing Medium. 6. Triturate the cells vigorously until a single-cell suspension is achieved. 7. Count the cells (see Protocol ID#1934). 8. Pellet the cells by centrifugation at 1000 rpm in a table-top centrifuge (800-1,000 X g) for 5 minutes. Aspirate the medium and resuspend the cell pellet in 20 ml of Chemotaxis Buffer. 9. Pellet the cells as in step 8. Aspirate the medium and resuspend the cells in Chemotaxis Buffer at 0.5 to 1 X 106 cells/ml. Maintain cells at 37°C until the chemotaxis chambers are ready to use. C. Preparation of Agonist Solution(s) 1. Dilute the agonists (chemokines or other chemoattractants) to the desired concentrations in at least 200 μl Chemotaxis Buffer (see Hint #6). Remember to include a negative control - Chemotaxis Buffer with no chemoattractant. 2. Degas the solution under vacuum for 10 minutes to minimize the formation of air bubbles underneath the filter during the experiment. |
首页
