Quantification of Maximum Invasive Depth

The depth of cell invasion is determined by measuring the distance from the top of the gel to the leading front of migrating cells using the calibrated micrometer present on the fine-focus dial of an inverted phase microscope (see Fig. 2). The leading front is defined as the point at which two of the leading cells within a given field are in the same focal plane under 200x magnification.

1. Measurements are taken in five fields within each well; using the center of the well as a landmark and as the first point (center point), the four additional measurements are taken in fields selected by moving the stage to points north, south, east, and west of center. The distance to each field is defined as half the distance from the center point to the edge of the well.

2. Starting near the bottom of the gel, turn the fine-focus dial so the focal plane moves toward the surface of the gel. When two cells appear in the same focal plane, begin counting revolutions until you reach the surface of the gel.

3. To calculate the distance from the gel surface to cells at the leading front, multiply the number of revolutions of the fine-focus dial by the number of ^m/revolution. For the Nikon Diaphot used in our lab, 1 revolution moves the focal plane 100 ^m.

3.2.3. Quantification of the Percentage of Invasive Cells

The percentage of invading cells is determined by using a limited trypsin and collagenase treatment to remove the noninvasive cells (i.e., cells attached at or near the gel

Proteoglycan Gel

Fig. 2. Measuring depth of cell invasion. Following the incubation of the cells on the collagen gels, the distance from the surface of the gel to the cells of the leading front is determined using the calibrated micrometer (on the fine-focus dial) of an inverted phase microscope. Illustrations of collagen gels with an invasive or noninvasive cell line are drawn on the left side of the figure. Photomicrographs taken of cells within collagen gels are shown on the right. In this example, for the invasive cell culture, invading cells are visible 300 ^m below the surface of the gel. However, for noninvasive cells, the field at 300 ^m below the surface of the gel is devoid of cells.

Fig. 2. Measuring depth of cell invasion. Following the incubation of the cells on the collagen gels, the distance from the surface of the gel to the cells of the leading front is determined using the calibrated micrometer (on the fine-focus dial) of an inverted phase microscope. Illustrations of collagen gels with an invasive or noninvasive cell line are drawn on the left side of the figure. Photomicrographs taken of cells within collagen gels are shown on the right. In this example, for the invasive cell culture, invading cells are visible 300 ^m below the surface of the gel. However, for noninvasive cells, the field at 300 ^m below the surface of the gel is devoid of cells.

surface). This is followed by complete digestion of the gel with collagenase and recovery of the invasive cells (see Fig. 3). Once the numbers of invading and no+ninvading cells are determined, the percentage of invading cells can be calculated.

1. Collection of the noninvasive cell population:

a. Aspirate the medium from the surface of each gel and discard.

b. Carefully wash the gel with 0.5 mL of PBS + 5 mM EDTA solution and collect by aspiration into the appropriate tube (see Note 12). Repeat this wash/collection step two additional times. Place all solutions (i.e., washes, trypsin, and collagenase solutions) collected from each well into the same 15-mL culture tube.

c. Incubate the collagen with 0.5 mL of trypsin/EDTA solution at room temperature for 30 min and collect the medium and released cells by aspiration. Although this digestion does not release many cells, it does prepare the collagen for the collagenase treatment.

d. Wash the gels twice and collect the solution two times as in step b.

e. Incubate the gels with 0.5 mL of collagenase solution (0.5 mg/mL of collagenase in 1 x RPMI, prewarmed to 37°C) for approximately 10 min at 37°C (see Notes 16-17).

f. Quickly but carefully remove the collagenase solution and wash the gels 3 times as in step b.

Diagram Collagen Seeding Theory

Fig. 3. Diagram of cell invasion assay. Collagen gels are prepared and seeded with cells. After the appropriate incubation period, first the depth of cell invasion is determined, followed by determination of the percent of cell invasion. Noninvading cells are recovered from the surface of the gels by trypsin and limited collagenase digestions and counted. Next, following complete digestion of the remaining collagen with collagenase, the invading cells are collected and counted. The percentage of cell invasion for each gel is calculated by dividing the number of invading cells by the total number of cells recovered from the collagen gel (noninvading + invading cells) and multiplying by 100.

Fig. 3. Diagram of cell invasion assay. Collagen gels are prepared and seeded with cells. After the appropriate incubation period, first the depth of cell invasion is determined, followed by determination of the percent of cell invasion. Noninvading cells are recovered from the surface of the gels by trypsin and limited collagenase digestions and counted. Next, following complete digestion of the remaining collagen with collagenase, the invading cells are collected and counted. The percentage of cell invasion for each gel is calculated by dividing the number of invading cells by the total number of cells recovered from the collagen gel (noninvading + invading cells) and multiplying by 100.

g. Centrifuge the tubes containing the noninvasive cell population at 300g for 10 min and resuspend the cell pellet in an appropriate amount of buffer for cell counting (i.e., hemocytometer or Coulter counter).

2. Collection of the invasive cell population:

a. Add 0.5 mL of collagenase solution to each well and incubate at 37°C until the gels are completely digested (approximately 1.5 h).

b. Collect the solution by aspiration into individual tubes. Place all solutions collected from each well into the same 15-mL culture tube.

c. Wash the wells and collect the solution by aspiration as in step b.

d. Centrifuge the cells and count as in g above.

3. Determine percentage of invasive cells:

invasive cells

- x 100 = % invasive cells noninvasive cells + invasive cells

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