- MB-series Chamber Differences
- Preparing the Chamber and Filling the Microplate Wells
- Filling the Top Wells
- Removing and Wiping the Filter
- Suggested Reading
NOTE: This protocol applies to all three MB-series 96-well chambers. The three chambers differ in top-well volume and diameter, as follows:
|Stock #||Well diameter (mm)||Upper Well Capacity
|Exposed Filter Area (mm2)|
* The MBC96 has a step in the upper well; capacity is 50µL if filled to the step, 225µL if filled to the top.
In this protocol, where it is helpful to distinguish among the three kinds of chambers, the features relevant to the MBA96, MBB96, and MBC96 will be listed in order, in brackets and separated by commas. For example the three volumes listed in the expression [395µL, 225µL, 50/225µL] belong to the upper wells of the MBA96, MBB96, and MBC96 respectively.
Preparing the Chamber and Filling the Microplate Wells
NOTE: MB series chambers are designed to be used with Neuro Probe disposable microplates, with Neuro Probe reusable 96 well inserts, and with a few microplates made by other manufacturers. Please see chemotaxis accessories for information about these microplates and inserts.
- Turn the two spring-loaded knobs on the top of the chamber counter-clockwise to release them, and flip the top plate of the chamber up to expose the bottom plate. Be sure the gasket is retained on the under-side of the top plate by the five stainless steel pins that are mounted in the plate (if the gasket is out of position, simply reposition it over the pins.).
- Whether you are using a recommended disposable microplate or a Neuro Probe reusable 96 well insert, you must first insert the appropriate spacer (see spacers), then place the microplate in the recess with the A1 well in the back left corner. If the plate does not fit snugly, place it against the A1 corner.
- Adjust a variable-volume micro pipette with a 1mm tip so that the ejected liquid fills a bottom well. The volume should be such that a slight positive meniscus forms; this prevents air bubbles from being trapped when the filter makes contact. Fill the bottom wells with chemoattractants or control reagents that have been warmed to about 37°C and vortexed to expel dissolved gases. To prevent excessive evaporation, keep the filling time under 10 minutes.
- Insert the filter into the bottom plate. Handle the filter frame by the edges only. Hold it over the bottom plate of the chamber with the filter side down, unless you are using Neuro Probe low volume plates (Stock# MP30). Place it against the aligning pins on one short side of the recess. Carefully lower it, making sure the filter is within the perimeter of the aligning pins around the recess. If you are using MP30 plates, be sure the holes in the corners of the frame go onto the pins in the corners of the plate. If you are using framed filters with adhesive, first remove the protective covering from the adhesive-backed frame, then position and insert the filter as described above. When the filter is in place, press down lightly on the frame to ensure it adheres to the bottom-well microplate. Again, handle the filter by the frame only — do not touch the filter membrane.
- Close the top plate and press down firmly on it as you turn the knobs about one-third turn clockwise to seal the plates together. It is important to maintain even, firm pressure on the top plate while you turn the knobs, as this prevents air bubbles from being drawn into the lower wells. Do not over-tighten the knobs.
NOTE: If the chamber seems to jam for any reason, do not force it closed. Check for debris which might be preventing proper closure. Be sure the microplate is seated in the recess properly and that the spacer is centered under the microplate. Check that the filter frame is within the perimeter of the aligning pins and that the gasket is properly installed over the five pins on the top plate. If the chamber still will not close easily, contact us to make arrangements to have the chamber serviced.
When the chamber is loaded and the top is closed but the knobs are not yet fastened, there is a gap of about 3mm between the front edges of the plates; this is normal. When the knobs are turned to secure the chamber, the top plate bows substantially; this maintains a seal across all the wells. When the chamber is properly assembled and the knobs are turned, the normal gap between the top and bottom plates is approximately 1.75mm at the midpoint of the short dimension of the chamber. Without a plate in the chamber, this gap diminishes to about 1mm.
Filling the Top Wells
Cell suspension for the top wells must be prepared at the appropriate concentration. The number of cells per milliliter in the cell suspension can be calculated, provided the optimum density of cells (cells per mm2) on the top side of the filter is known. The exposed filter area of the site in mm2 multiplied by the cells per mm2 will give the optimum number of cells per site, and that number along with the volume of cell suspension to be placed at each site will give the number of cells per milliliter of media in the suspension.
The optimum density of cells on the filter must be determined as an integral part of the design of the chemotaxis assay. Please see cell-activity assays for a discussion of this process.
- Pipette cell suspension into each top well, filling the wells so that a slight positive meniscus is formed. (If you are using the MBC96 model, fill to the step in the top well with 50µL of suspension, using a 1mm pipette tip. To use the entire 225µL of the top wells, let the cells in the initial 50µL settle, then add 175µL of media without cells to fill the wells.) To avoid trapping air bubbles, hold the pipette at a steep angle, the pipette tip should be deep in the well, resting against the well wall just above the filter , while the side of the pipette tip should rest against the top rim of the well.
- Check for trapped bubbles in the wells. One test is to look at the reflections of overhead lights in the meniscuses of the wells: an abnormally large positive meniscus usually means a trapped air bubble. To remove bubbles, suck the well completely dry with a suction line and disposable pipette tip, then refill the well. Residual fluid in the top wells can also be removed with a cotton swab.
- For most chemotaxis assays the filled chamber is incubated at 37ºC in humidified air with 5% CO2. Incubation time depends on the type of cell and the chemotactic factors. See incubation time for ways to determine optimum incubation periods.
Removing and Wiping the Filter
- Aspirate fluid from the top wells or empty them by shaking the chamber over a sink or container.
- Place the chamber on a flat surface and open it as described in the first section of this protocol. Important: While turning the knobs and opening the chamber, press down on the filter frame through the cutouts in the ends of the top plate to ensure that the filter remains with the microplate when the top plate is raised. (If you are using filters with adhesive, the filter will be bonded to the plate.) If the gasket has dislodged and is clinging to the filter rather than to the top plate, remove it carefully.
- Using the semi-circular recesses at the front and back of the bottom plate for your thumb and forefinger, lift the microplate/filter assembly straight up. Handle the assembly — and the filter and microplate separately — by the edges only; do not touch the flat surfaces.
- Immerse the top and bottom plates, with hardware and gasket, in cool distilled water. Never allow solutions to dry on reusable chamber components. Please review the warranty and the cleaning and sterilizing instructions. (If you are using a reusable 96 well insert, follow the same procedure after quantification.)
- Holding the filter or filter/microplate assembly by the edges only, gently wipe the nonmigrated cells off the top of the filter with a cotton swab, cell harvester, or small squeegee. Hold the unit at 45 degrees over a sink or container and carefully flush the top surface of the filter with cell-suspension media or PBS. Apply the rinse to the top edge of the filter and allow it to gently flow across the filter surface. The goal is to remove non-migrated cells from the top surface without disturbing cells that have migrated through the filter.
- If you are using filters with adhesive and plan to count cells in the wells of the microplate, place the filter/microplate assembly in a microplate centrifuge and spin the assembly to move migrated cells from the bottom side of the filter into the wells of the microplate. The speed and length of time necessary to accomplish this will vary depending on the type of cells you are using.
Shi, Kornovski, Savani, and Turley. “A Rapid, Multiwell Colorimetric Assay for Chemotaxis.” 1993, Journal of Immunological Methods, 164, 149-154.
Junger, Cardoza, Liu, Hoyt, and Goodwin. “Improved Rapid Photometric Assay for Quantitative Measurement of PMN Migration.” 1993, Journal of Immunological Methods, 160, 73-79. [NOTE: the Neuro Probe chemotaxis chamber illustrated in this paper is an early prototype of the MB series instruments; it has no hinges or spring-loaded catch.]