Preparing the Chamber
- Place a filter on the filter seat of a clean, dry chamber and screw in the retainer by hand; do not over-tighten the retainer, as this could cause the filter to tear. Do not touch the filter with your fingers; lipids on your skin can act as chemotactic factors and may alter assay results.
NOTE: It is important to equalize the fluid levels in the top and bottom wells as quickly as possible so that hydrostatic pressure will not cause movement of fluids through the filter. To equalize levels, fill the upper and lower wells as rapidly as possible.
- Adjust a variable-volume micropipette so that the fluid expressed will fill the lower well of the Boyden chamber up to the plug — about 1,175 µL. The chemotactic factor solution (or control solution, if the chamber is functioning as a negative control) should be warm and de-gassed to prevent bubbles appearing during incubation.
- Tilt the chamber and fill the lower well to the plug. Be careful not to leave bubbles that may get trapped beneath the filter.
- Quickly right the chamber and check that the solution has not flooded the upper well. If flooding occurs, the filter is faulty, there is debris on the filter seat, or the contact surface of the chamber is damaged. Remove the filter, inspect the sealing surfaces, and begin again with a new filter. If the problem persists, put a drop of washable ink on the contact surface of the chamber and screw down the filter retainer. If the ink forms a uniform ring all around the surface where the retainer contacts the chamber, the chamber and the retainer are not at fault, and the problem lies with the filters or in the filling technique.
Preparing and Adding Responding Cells
- The concentration of cells in the suspension should be adjusted so that 560 µL contains the desired number of cells for the well. Since the exposed filter area in the well is 50 mm2, a suspension of 50,000 cells in 560 µL will yield 1,000 cells/ mm2 of filter area.
- Fill the upper well (in the filter retainer) with 560 µL of cell suspension. Hold the pipette at an angle so that the end of the pipette tip rests against the wall of the well just above the filter. Eject the fluid with a rapid motion to dislodge air in the bottom of the well.
- When the top well is full, fill the bottom well to the rim of the vertical hole with additional chemotactic, or control, solution.
- Incubate the chamber. For most chemotaxis assays this is done at 37°C in humidified air with 5% CO2. Optimum incubation times vary considerably depending on cell types and chemotactic factors. See incubation time for a method of determining the optimum time.
Removing and Staining the Filter
- After incubation carefully remove the fluid from above the filter and from the bottom well. Unscrew the retainer and lift the filter out with forceps.
- Immerse the retainer and bottom well in cool distilled water. See cleaning and sterilizing instructions for proper care of the instrument.
- If your assay requires that nonmigrated cells on top of the filter be removed before the migrated cells are stained, gently wipe the nonmigrated cells off with a cotton swab. Wipe twice, using two swabs or both ends of a double-tipped swab.
- Stain the migrated cells on the bottom of the filter and count under a microscope.
NOTE: These instructions assume that you are working with polycarbonate filters. If you are working with cellulose nitrate filters, please read the protocols for staining cellulose nitrate filters.