- General Description
- A Visual Assay Using the Z02, Courtesy of Sally Zigmond
- Notes and Warnings
After a general description of the process of using a Z02 chamber, a specific protocol describing the chamber’s use in a particular type of experiment will be given.
In general a drop of suspended cells is placed on the middle of the cover glass. After the cells have adhered, the ends of the cover glass are wiped dry, leaving a 6mm-wide wet section with adhering cells.
The cover glass prepared in this way is inverted onto the grooved microscope slide so that the cell-covered portion is centered over the bridge between the grooves. The clamps are installed, and cell-suspension media and chemotactic factor are pipetted into the two grooves until they are full and no bubbles remain.
The chamber is then incubated.
After a suitable period (depending on cell type and experiment goals) the slide is placed under a microscope and the orientation of cells is observed and quantified. The internal structures of the migrating cells can also be studied by using high-power light microscopy.
A Visual Assay Using the Z02, Courtesy of Sally Zigmond
- Clean the chamber before each use. Wash it in warm water with tissue culture detergent, rinse well with distilled water and wipe dry. The bridge may also be wiped with 70% or 95% ETOH.
- Place a drop of blood (e.g. from a finger prick) across the center of a 22mm x 40mm cover glass. Enough blood must be placed on each cover glass to allow it to clot and partially retract without drying. It takes close to 100 µL per cover glass.
- Place the cover glass with blood in a moist chamber (e.g. a petri dish containing a wet piece of filter paper) at 37°C, with or without CO2.
- After about 45 minutes the blood clots and begins to retract, and fluid is visible around the edges. At this point the clot and red blood cells can be gently rinsed off with 0.9% saline. A monolayer of cells, mostly neutrophils, remains on the cover glass. Do not let the cells dry.
- Prepare a 10% gelatin stock by dissolving gelatin in boiling de-ionized water. Warm the stock to melt, and dilute it 1:10 with Hanks medium in which the bicarbonate has been removed and replaced with HEPES buffer (2.40 g HEPES/liter) at pH 7.2. Make sure the gelatin is really in solution when diluting with the Hanks. This medium is slightly acidic and slightly hypotonic. These conditions contribute to good cell locomotion; alkaline pH and hypertonicity are inhibitory.
- Rinse the cell layer on the cover glass with a few drops of this incubation medium. Quickly drain off the fluid on the cover glass, dry the ends of the cover glass with a Kimwipe, and invert the cover glass onto the chamber so that the cells lie over the bridge.
- Place the clamps on each side of the cover glass-microscope slide assembly. Do not move the cover glass; any movement will lyse the cells on the bridge. The chamber assembly requires a little practice. With a minimal amount of fluid over the cells (but not allowing the cells to dry) the distance between the cover glass and the bridge will be very thin; 5 microns is optimal (the cells will appear slightly squeezed). If this gap is too large, the cells will not orient well. The gap can be measured by the difference in focal plane between the top of the bridge and the bottom of the cover glass, using the micrometer on the fine-focus knob of the microscope. With practice you can lower the cover glass from one side. This helps eliminate air bubbles on the bridge; they can interfere with cell orientation.
- After the cover glass is secured, pipette 100 µL of media into an open end of one of the grooves. Pipette 100 µL of chemotactic factor (or cell-suspension media for a negative control chamber) into the other groove.
- Incubate at 37°C for about 20 minutes to allow the cells to respond. (For other cell types longer incubation may be desirable.)
- Use a 40X-phase objective to observe the cells on the bridge and evaluate cell orientation. Orientation can be scored by the cell morphology. The front of a locomoting cell has a broad lamellipodium, while the tail is thinner and can be knob-like or drawn out into retraction fibers. If the chamber cools at room temperature for awhile, the cells round up and are more difficult to score. Orientation is easiest to score when the cells are moving well.
- Orientation should be scored at a specific location on the bridge — the chemoattractant side, the middle, or the media side. The level of orientation can vary greatly across the width of the bridge, depending on the concentration of chemoattractant. Scan a given chamber along the bridge until at least 100 cells have been scored.
A number of artifacts can alter the level of orientation seen in a given field.
- Cells adjacent to air bubbles and cell clumps tend to be idiosyncratic; these factors modify the effects of the chemical gradient.
- If cells do not develop polarized morphology, they may be dead on the bridge but alive over the grooves. This can be caused by a dirty bridge, or the prep may be so thin that the cells are actually lysed.
- If cells develop polarized morphology but do not orient, check to see whether the gap between the bridge and the cover glass is thin enough. If the gap is appropriate, try a different concentration of chemoattractant.
Notes and Warnings
Zigmond glass slide chambers are fragile and easily broken, so handle and use with care. The clamps that retain the cover glass have pins that fit into holes in the microscope slide. The fit is snug, so to remove you may have to rotate the knob clockwise as you lift up on the clamp. Never try to remove the pins by prying.
A small Allen wrench is provided with the chamber. If the pins do not twist out fairly easily, turn the chamber over, insert the wrench in the hexagonal hole on the bottom of the pin, and turn it. Turning the pins, as opposed to pulling them, minimizes the chance of chipping the glass.