Preparing the Wells
The Neuro Probe three tiered chamber has three precision-machined acrylic plates: a clear bottom plate with hardware holes only, a middle plate with 48 through holes and six permanently installed hardware posts, and a top plate with hardware holes and 48 through holes. The chamber is assembled with silicone gaskets separating the plates. When assembled, the bottom and middle plates form the bottom wells.
- Place the middle plate on the work surface so that the NP trademark and serial number are reverse reading. Place a gasket over the six hardware posts so that the diagonally cut corner of the gasket is near the trademark. If your assay calls for a filter or a membrane covering the bottom of the bottom wells, cut off one corner of the membrane and position it over the gasket, aligning the angled corner with the trademark and centering the membrane so that it covers the 48 holes.
- Place the bottom plate on top of the gasket and membrane (if any) so that the NP trademarks on the two plates are aligned. Apply thumbnuts to the six threaded posts and finger-tighten firmly to fasten the plates together. Do not use pliers or a wrench as this can strip the threads. Turn the assembly over and place on work surface with the bottom plate down. The 48 middle-plate holes now form 48 bottom wells.
- 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. Use a variable-volume micropipette with a 1mm tip and adjust the volume so that the ejected liquid fills one well. The wells will hold about 50µL. A slight positive meniscus should form when the well is filled; this prevents air bubbles from being trapped when the filter is applied.
- Cut 1mm off the corner of a filter and orient it with the cut corner towards the NP trademark on the middle plate. Lift the filter by the ends with two pairs of forceps, hold it 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 too much movement will cause cross contamination between wells.
- Apply the second gasket with its angled corner over the NP trademark of the middle plate. Slide the gasket part way down on the six hardware posts, but do not push it down onto the filter.
- Orient the top plate so that its NP engraving is positioned over the trademark on the middle plate. Firmly press the top plate down onto the middle plate with the gasket and filter sandwiched between the two plates. Do not release the pressure. While still firmly holding the plates together, apply the six remaining thumbnuts to the threaded post hardware. Release pressure to the plate only when all six thumbnuts are evenly and firmly tightened. Again, do not use pliers or other tools.
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. Although different volumes can be used, 50µL just fills the wells and forms a slight positive meniscus, which facilitates inspection for bubbles. 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.
- It is essential to avoid trapping air bubbles in the upper wells. To check for trapped air bubbles, look at the reflections of overhead lights in the meniscuses: a well with an abnormally large positive meniscus usually has a trapped air bubble. Remove any bubbles by sucking 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 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.
- If there is no membrane on the bottom of the bottom wells, an inverted microscope can be used to view nonadherent migrated cells through the clear 5.7mm-thick bottom plate of the A3BP48. If the microscope has a long working distance, adherent cells can also be observed on the under side of the filter another 5.7mm above that. Or the chamber can be cytocentrifuged to spin all migrated cells to the bottom of the bottom wells where they can be viewed through the bottom plate.
- If a membrane has been placed at the bottom of the bottom wells, the chamber can be cytocentrifuged and the bottom membrane can be removed and stained to count all migrated cells: Remove the thumbnuts from the bottom of the chamber and place on the work surface with the bottom plate down. Lower the bottom plate evenly so that it stays level as it drops to the work surface. With forceps keep the gasket with the middle plate and posts, while the membrane moves down with the bottom plate. Remove the middle and top plates.
- Lift up one end of the membrane with forceps and catch 1mm of the edge in the wide filter clamp. Lift the membrane and quickly attach the small filter clamp to the edge of the free end.
- For granulocytes and monocytes, carefully immerse the filter membrane in methanol, and then place it cell-side up on a disposable lint-free towel for air-drying.
- During all procedures, as parts of the chamber are removed put them in cool distilled or de-ionized water to soak.
- When a filter membrane is dry, clamp the edge of one end with a wide clamp, weight the other end with the small clamp and stain in Diff-Quik (by RAL Diagnostics, available from many major laboratory suppliers) 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. See Chemotaxis Accessories.
- Place the wet filter membrane 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.
- Follow the instructions for cleaning and sterilizing your A3BP48 chamber. Reread and heed the warnings.
- To count migrated cells adhering to the under side of the filter that forms the bottom of the top wells, remove the thumbnuts from the top plate but keep it from coming loose as you invert the entire chamber onto a paper towel. Keep the top plate level as it moves down to the work surface, taking the gasket and filter with it. Remove the middle and bottom plates. 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 wide 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 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, wet the underside of the filter again in PBS, and repeat Step 11. Clean the wiper again, wet the filter a third time in PBS, and repeat Step 11.
- Dry and stain the filter as in Steps 4 through 8 above.
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.