NOTE: The AC48 is designed for cellulose nitrate filters. Polycarbonate filters may also be used in the chamber and may either be held by the retaining hardware as described here for cellulose filters, or placed directly over the filled bottom wells with forceps. If you are using polycarbonate filters, please see staining polycarbonate filters for instructions. You may want to purchase an accessory pack (Stock # P48AP), which includes forceps, filter clamps, and a wiper designed to manipulate and process polycarbonate filters.
- Assembling and Filling the Chamber
- Preparing and Adding Responding Cells
- Removing the Filters
- Suggested Reading
Assembling and Filling the Chamber
- Adjust a variable-volume micropipette with a 1mm tip so that the ejected liquid fills a bottom well (approximately 25µL). The volume should be such that a slight positive meniscus forms, so that no air bubbles are trapped when the filter is applied.
- Orient the bottom plate on a flat surface so that the NP trademark is at the upper right. 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 five minutes to prevent excessive evaporation.
- Orient the top plate, top down, with the NP trademark at the upper left. Notice that the silicone gasket is held against the top plate by eight button-head screw heads.
- Wet two 34mm x 41mm pieces of cellulose nitrate filter with medium; this can be done either before or after you position the filters. Place the filters so that they lie flat against the gasket. Tuck each filter edge under the screw heads to hold the filter in place.
- Turn the top plate assembly over and place it on the bottom plate, aligning the NP trademarks on the top and the bottom plates. 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 thumbnuts until finger tight. Do not use pliers or other tools to tighten them.
- You may remove the gasket for cleaning by carefully stretching it over the heads of the screws. When you reapply it, be sure that the screw heads come completely through the holes in the gasket.
- If you are using an AC48 template (Stock # C48TM) to help locate cell sites on the filters, modify these steps according to the C48TM 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.
- Pipette cell suspension into each upper well, adjusting the volume so that the filled well has a slight positive meniscus (note that it is essential to avoid trapping bubbles in this step). 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, and 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 after a set period (e.g. 30 minutes), and remove the rest sequentially, one every five 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.
Removing the Filters
- After incubating the chamber, aspirate fluid from the top wells or empty them by shaking the chamber over a sink or container.
- After emptying the upper wells, remove the thumbnuts while holding down the top plate. Invert the entire chamber onto a paper towel. Grasp the four corners of the top plate and push down evenly so that the top plate stays parallel to the bottom plate as it drops to the table. Remove the bottom plate and immerse in cool distilled or de-ionized water.
- Using forceps, slip the filter edges from under the heads of the retaining hardware and lift the filters off the top plate. Immerse the top plate in cool distilled or de-ionized water.
- Clip the filters to a 50 x 75mm glass slide for staining. You can also cut the filters and use 25 x 75mm glass slides. We recommend using stainless steel clips (Stock # SSC4). The clips can be used either singly or in pairs for each filter, securing the filter either at the ends or in the middle. In either method it is important that fixing and staining solutions can permeate between the filter and the slide.
- Follow the instructions for staining cellulose nitrate filters. (If you are using polycarbonate filters, see the instructions for staining polycarbonate filters.)
- Follow the instructions for cleaning and sterilizing your AC48 chamber. Reread and heed the warnings.
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.
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.
Richards and McCullough. “A Modified Microchamber Method for Chemotaxis and Chemokinesis.” 1984, Immunological Communications, 13 (1), 49-62.
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.