Neuro Probe A-Series (AA96, AB96, AC96)

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

NOTE: This protocol applies to all three A-series 96-well chambers. The three A-series chambers differ in well volume and diameter, as follows:

Stock #	Well diameter (mm)	Upper Well Capacity (µL)	Lower Well Capacity (µL)	Exposed Filter Area (mm²)
AA96	6.4	390	80	32
AB96	4.8	225	30	18
AC96	3.2	50/225*	25	8

* The AC96 has a step in the upper well; capacity is 50µL if filled to the step, 225µL if filled to the top.

Where it is helpful to distinguish among the three kinds of chambers in this protocol, the features relevant to the AA96, AB96, and AC96 will be listed in order, in brackets and separated by commas. For example the three volumes listed in the expression [80µL, 30µL, 25µL] correspond to the lower-well volumes of the AA96, AB96, and AC96 respectively.

Preparing the Chamber and Filling the Bottom Wells

1. On a flat surface unscrew and remove the eight thumbnuts and separate the two plates. The gasket will adhere to the top plate. If the gasket sticks to the bottom plate, making it difficult to separate the plates, grasp the bottom plate firmly and lift one corner of the top plate; this should free the entire plate, with gasket attached.
2. Invert the top plate and place it on a clean paper towel with the gasket facing up. Orient the top plate so that the NP trademark is in the upper right corner with the letters wrong-reading. The top-plate registration pins will come up through matching holes in the gasket.
3. Orient the bottom plate so that the NP is right-reading at the upper left; the numbers and letters that label the grid of wells should also be right-reading.
4. Adjust a variable-volume micropipette with a 1mm tip so that the ejected liquid fills a bottom well. The well will hold about [80µL, 30µL, 25µL]. Pipette enough fluid to form a slight positive meniscus; this should prevent air bubbles from being trapped when the filter is applied.
5. Warm chemoattractants or control reagents to about 37ºC and de-gas them by vortex or vacuum. Use the adjusted micropipette to fill the bottom wells, completing the 96 wells in no more than 10 minutes to prevent excessive evaporation.
6. Attach the framed filter to the top plate, referring to A Series Figure 1 for this step. Do not touch the filter membrane with your fingers or with any instruments that may contaminate it; lipids on your fingers can act as chemotactic factors for cells. The framed filter is designed to fit on the top plate in one way only. The frame is asymmetric and is designed to fit correctly on the top plate in one way only. (see A Series Figure 1). Turn the filter over, find the letter N on the frame and orient the frame so that the letter N is on the left side. Next to the letter N on the outside edge of the frame is a small curved indent which must align with the pin marked B. Gently push the frame against the pair of pins (C) on the long edge of the top plate. The indent should be aligned with pin (B). If it is not in alignment, then align the indent with pin (B). Push down on the frame so that the filter is touching the gasket. (This illustration is currently being edited.)

With the framed filter installed, invert the top plate onto the filled bottom plate. Orient so that the NP trademarks on the two plates line up. Avoid trapping air bubbles between the filter and the fluid in the bottom wells by pressing down evenly on the middle of the long edges of the top plate (between the hardware posts) while you tighten the thumb nuts. Do not let the top plate rock. Tighten the nuts gradually and alternate between edges rather than tightening all four nuts on one edge and then the nuts on the other edge.

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.

1. Pipette cell suspension into each top well, filling the wells so that a slight positive meniscus is formed. (If you are using the AC96 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; as illustrated in A Series Figure 2, the pipette tip (2A) should be deep in the well, resting against the well wall just above the filter , while the side of the pipette tip (2B) should rest against the top rim of the well. (This diagram is currently being updated)

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.
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 methods of determining optimum times for your experiment.

Removing, Wiping, and Staining the Filter

Aspirate fluid from the top wells or empty them by shaking the chamber over a sink or container.
Remove the thumb nuts while holding down the top plate, then remove the top plate, invert it, and place it on a clean paper towel. Alternatively, invert the entire chamber onto a paper towel, grasp the 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 work surface. Lift the bottom plate off.
Lift up on the framed filter from the single pin (B) side (see Fig. 1) to release it, and lift it off the top plate.
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.
Various methods can be used to count the cells. In the simplest, the cells should be tagged with a fluorescent tag before incubation.
Holding the filter 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 media, water, 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.
Place the filter in a fluorescence-type microplate reader as you would a 96 well microplate. Always put the reference corner of the framed filter (see A Series Figure 2) away from any springs in the plate holder. Count cells following the protocol for your reader.
You may wish to fix and stain the adherent migrated cells to be counted visually. To fix the cells, place the framed filter cell-side-down in a shallow dish of methanol or other fixative and soak in accordance with the instructions provided by the fixative manufacturer. Do not let the fixative run onto the non-cell side of the filter.
To stain the adherent migrated cells, aspirate the fixative out of the dish and replace it with Diff-Quick (RAL Diagnostics), Leukostat (Fisher), or equivalent dye. Allow the filter to air dry completely before counting migrated cells.

Preparing the Chamber and Filling the Bottom Wells

Filling the Top Wells

Removing, Wiping, and Staining the Filter

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