Preparing the Chamber
- Adjust a variable-volume micropipette so that the fluid expressed will fill the lower well of the blind well chamber.
- Fill the clean, dry lower well with chemotactic reagents or a control
solution. A slight positive meniscus should form when the well is
filled; this helps prevent air bubbles from being trapped when the
filter is applied.
- With vacuum tweezers or small forceps, place a filter over the
filled well, being careful not to trap air bubbles. (A Pasteur pipette
with a tube for mouth suction will work in place of vacuum
tweezers.) Do not touch the filter with your fingers; the lipids on
your fingers can act as chemotactic factors and alter assay results.
- Screw in the filter retainer by hand; do not use wrenches, pliers, or other tools.
Preparing and Adding Responding Cells
- The concentration of cells in the suspension should be adjusted so that
the volume of suspension to be placed in the upper well contains the
desired number of cells for the well. For example, suppose you intend
to fill the upper wells to the top — with 200 µL of cell
suspension (800 µL if you are using a BW200L chamber). Since
the exposed filter area for each well is 18 mm2 (50 mm2 for the BW200L), a suspension of 18,000 cells in 200 microliters will yield 1,000 cells/ mm2 of filter area. (In the BW200L 50,000 cells in 800 microliters will yield approximately 1,000 cells/mm2 of filter area.)
- Pipette cell suspension into the upper well, which is in the retainer.
it is not necessary to fill to the top. 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.
- Incubate the filled 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.
- Unscrew the retainer and immerse it in cool distilled water.
- Lift the filter out with forceps and immerse the 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.
These directions 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.