NOTE: The following 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.
- Preparing the Chamber
- Preparing and Adding Responding Cells
- Staining Polycarbonate Filters
Preparing the Chamber
- Adjust a variable-volume micropipette with a 1mm tip so that the ejected liquid fills a bottom well. The well will hold 25 to 26 µL. A slight positive meniscus should form when the well is filled; this prevents air bubbles from being trapped when the filter is applied.
- Orient the bottom plate on a flat surface so that the NP trademark is at the upper left. Warm chemoattractants or control reagents to about 37°C and de-gas them by vortex or vacuum. Fill the bottom wells, completing the 48 wells in no more than 5 minutes, to prevent excessive evaporation.
- Cut 1mm off the corner of a filter membrane and orient it with the cut corner at the upper left. Lift the filter by the ends with two forceps, hold it evenly over the filled wells, and lower it onto them, allowing the middle portion of the filter to make contact first. The filter position can be adjusted at this point if necessary, but note that too much movement will cause contamination between wells.
- Apply the silicone gasket with its cut corner at the upper left, then the top plate, aligning its NP trademark with the trademark on the bottom plate. Push the top plate down firmly and do not let go; this helps prevent air bubbles from being drawn in and trapped in the bottom wells. With your free hand, apply and tighten the thumb nuts until finger tight. Do not use pliers or other tools to tighten them.
- If you are using an AP48 template (stock # P48TM) to help locate cell sites on the filters, modify these steps according to the P48TM protocol.
Preparing and Adding Responding Cells
- In the upper wells the concentration of cells in the suspension should be adjusted so that 50µL contains the desired number of cells for one well. For example, since the exposed filter area for each well is 8mm2, a suspension of 8,000 cells in 50µL will yield 1,000 cells/mm2. 50,000 cells in 50µL will yield approximately 6,000 cells/mm2.
- Adjust the pipette volume so that the filled wells have a slight positive meniscus and pipette cell suspension into each upper well. Hold the pipette at a steep angle so that the end of the pipette tip rests against the wall of the chamber just above the filter, and the side of the tip rests against the top rim of the well. Eject the fluid with a rapid motion to dislodge air in the bottom of the well.
- Check for trapped bubbles in the upper wells. One easy way to do this is to look at the reflections of overhead lights in the meniscuses: a well with an abnormally large positive meniscus usually has a trapped air bubble. To remove any bubbles, suck the well completely dry with a suction line and disposable pipette tip, then refill it.
- For most chemotaxis assays the filled chamber is incubated at 37°C in humidified air with 5% CO2. Incubation times vary considerably depending on cell types and chemotactic factors. One good way to determine the optimum incubation time is to use 6 to 12 blind-well chambers (e.g. stock # BW100) set up as negative controls and placed simultaneously in the incubator. Remove one blind-well chamber after a set period (e.g. 30 minutes), and remove the rest sequentially, one every 5 minutes. Stain the filters and examine them to see how long unstimulated cells have taken to migrate through the filter, or, if you are using cellulose nitrate filters, to a specified optimum depth. See incubation time for more information.
Staining Polycarbonate Filters
- Aspirate fluid from the top wells or empty them by shaking the chamber over a sink or container.
- Remove the thumbnuts while holding down the top plate, and invert the entire chamber onto a paper towel. Grasp the four corners of the top plate (now on the bottom) and push down evenly so that it stays level as it drops to the table. If the gasket hangs up on the post hardware, carefully push it down evenly onto the plate. Take care not to touch the filter, which should be stuck to the gasket. Immerse the remaining plate (with stud hardware in place) in cool distilled water.
- The migrated cells are now facing up on the filter, this side of the filter is henceforth referred to as the cell side. Lift up one end of the filter with forceps and catch 1mm of the edge in the large filter clamp. Lift the filter and quickly attach the small filter clamp to the edge of the free end. Keeping the cell side up, wet the underside (non-migrated cell side) of the filter in a dish containing PBS. Do not let the PBS wash over the cell side of the filter.
- Holding the filter by the large clamp, with the small clamp attached to the other end and hanging free, wipe the cells off the non-migrated cell side of the filter by drawing the filter up over the wiper blade. The blade should first contact the filter just below the jaws of the wide clamp. Use only gentle pressure against the blade, and maintain an angle of about 30° from the vertical for the portion of the filter above the wiper. It is important to complete the wiping carefully and quickly so that the cells will not dry on the filter; drying takes place in 10 to 20 seconds, and will prevent complete removal of the non-migrated cells.
- Clean the wiper with a cotton swab, again wet the underside of the filter in PBS, and repeat Step 4. Clean the wiper again, then wet the filter a third time in PBS, and repeat Step 4.
- For granulocytes and monocytes, carefully immerse the filter in methanol, then place the filter cell-side up on a disposable lint-free towel for air-drying. Rinse all chamber components in cool distilled water. For other kinds of cells, consult the literature for staining techniques.
- When the filter is dry, clamp the edge of one end with a large filter clamp, weight the other end with a small filter clamp, and stain in Diff-Quik® or equivalent dye, according to the manufacturer’s instructions. To avoid contaminating the chamber components with stain, it is convenient to have two sets of filter clamps, one for removing the filter from the gasket, and one for staining.
- Place the wet filter cell-side up on a 50 x 75mm microscope slide to dry. When the filter is dry, center it on the slide and place a drop of immersion oil on it. Rub the oil over the filter with a smooth, blunt instrument to remove all bubbles and wrinkles. The filter is now ready for counting.
- If you are using an AP48 template to help locate cell sites on the filter (stock # P48TM) modify these steps following the P48TM protocol.
AP48_protocol for Using Neuro Probe Reusable Multiwell Chemotaxis Chambers (PDF)
Falk, Goodwin, and Leonard. “A 48 Well Micro Chemotaxis Assembly for Rapid and Accurate Measurement of Leukocyte Migration.” 1980, Journal of Immunological Methods, 33, 239-247.
Falk, Harvath, and Leonard. “Only the Chemotactic Subpopulation of Human Blood Monocytes Expresses Receptors for the Chemotactic Peptide N-Formylmethionyl-Leucyl-Phenylalanine.” 1982, Infection and Immunity, 450-454.
Gnessi, Ruff, Fraioli, and Pert. “Demonstration of Receptor-mediated Chemotaxis by Human Spermatazoa.” 1985, Experimental Cell Research, 161, 219-230.
Harvath, Falk, and Leonard. “Rapid Quantification of Neutrophil Chemotaxis: Use of a Polyvinylpyrrolidone-free Polycarbonate Membrane in a Multiwell Assembly.” 1980, Journal of Immunological Methods, 37, 39-45.
Harvath and Leonard. “Two Neutrophil Populations in Human Blood with Different Chemotactic Activities: Separation and Chemoattractant Binding.” 1982, Infection and Immunity, 36 (2), 443-449.
Richards and McCullough. “A Modified Microchamber Method for Chemotaxis and Chemokinesis.” 1984, Immunological Communications, 13 (1), 49-62.