Reusable Chemotaxis Chamber Protocols > Single-well Chambers > Single Blind well chambers

Neuro Probe BW25, BW100, BW200S, and BW200L
Blind Well Single Well Chemotaxis Chambers Protocol

Please read the warranty and the cleaning and sterilizing instructions. Solvents, autoclaving, and improper maintenance will damage these acrylic instruments.

• Preparing the Chamber
• Preparing and Adding Responding Cells
• Removing and Staining the Filter

Preparing the Chamber

1. Adjust a variable-volume micropipette so that the fluid expressed will fill the lower well of the blind well chamber.
2. 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.
3. 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.
4. Screw in the filter retainer by hand; do not use wrenches, pliers, or other tools.

Preparing and Adding Responding Cells

1. 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 microliters of cell suspension (800 microliters 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.)
2. 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.
3. 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

1. After incubation carefully remove the fluid from above the filter.
2. Unscrew the retainer and immerse it in cool distilled water.
3. 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.
4. 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.
5. Stain the migrated cells on the bottom of the filter and count under a microscope.

NOTE: 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.