Incubation Time for Neuro Probe Instruments

A loaded chemotaxis instrument has cells suspended in media on top of a filter membrane; the cells respond to chemoattractants in the wells beneath the filter. The instrument is incubated to allow the cells to migrate through the filter membrane.

Some cells migrate through the filter even though they are not stimulated to move along a concentration gradient of a chemoattractant. This is called random migration. The rate at which random migration occurs can be tested by setting up a series of negative controls, or filter sites at which cell suspension is placed on top of the filter, but the corresponding bottom well contains only cell-suspension media, no chemoattractant. (There is a general discussion of different kinds of controls in cell migration assays at controls.)

The loaded instrument is incubated long enough for many stimulated cells to migrate but not long enough for significant random migration in either test wells or negative control sites. The optimum incubation time is the time at which the differential is highest between the number of stimulated cells and the number of unstimulated cells that have migrated. Typically this is the time when unstimulated cells at negative control sites begin appearing in the bottom well and as many stimulated cells as possible have had time to migrate, but the number of migrated unstimulated cells is close to zero.

There are a number of ways to determine the optimum incubation time. Two methods are given here; both assume that the optimum time is approximately 45 minutes. Based on the literature and previous experiments, you must estimate the optimum time for the cells and reagents in your assay and adjust the times accordingly in the methods outlined below.

Method 1 works with multiwell instruments while Method 2 is preferred if you have single well chambers available.

Method 1

  1. Suppose a 96 well instrument is being used. (This method can be adapted for 48 well chemotaxis chambers as well.) The filter sites will be in an 8 x 12 array of sites with rows labeled A through H and columns labeled 1 through 12. Negative-control sites and sites with chemoattractant can be distributed in a pattern to exhibit the maximum differential after analyzing the cell counts on the filter, in the bottom wells, or both. The following is one example of such a pattern.
  2. In the microplate (or bottom wells) fill odd-numbered columns with chemoattractant solution and even-numbered columns with media but no chemoattractant. Attach the filter (and top plate if using a reusable chamber) and pipette cell suspension onto the filter sites of columns 1 and 2. Cover with the clear plastic lid if using a ChemoTx instrument, and place in the incubator for five minutes.
  3. At five-minute intervals remove the instrument from the incubator, pipette cell suspension in the next two columns of the filter array, and return the instrument to the incubator for another five minutes.
  4. When cell suspension has been added to columns 11 and 12, incubate for 30 minutes.
  5. Remove the instrument from the incubator. Column 1 now contains eight sites with stimulated cells incubated for 55 minutes. Column 2 contains eight negative-control sites incubated for 55 minutes. Column 3 contains stimulated cells and column 4 contains negative controls, both incubated for 50 minutes. This pattern continues across the array to column 11 with stimulated cells and column 12 with negative controls, both incubated for 30 minutes.
  6. When cells are counted for the entire array of sites, analysis of the results should show where the maximum differential between negative controls and stimulated-cell sites lies. (Assuming the optimum incubation time lies between 55 and 30 minutes. The procedure must be adapted to span the estimated optimum time for your cells and reagents.)

Method 2

  1. Prepare 6 to 12 single well chemotaxis chambers (e.g. Stock # BW100) as negative controls: put cells suspended in media in the upper wells and media alone in the bottom wells.
  2. Place all the negative-control chambers in the incubator at the same time, and then remove them one by one at fixed intervals (e.g. one every 5 minutes, after an initial incubation of 30 minutes). Stain and examine the filters.
  3. The filters form an incubation-time series, and any cells that pass through the filters are unstimulated cells that have randomly migrated. Therefore the first filter in the series that shows cells on the bottom side represents a good approximation of the optimum incubation time.
  4. To verify this approximation, do a time course experiment with both positive and negative controls as described in Method 1.