Method of Measuring Pit Area


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Procedures

1. Plate Washing

(1)    Aspirate the culture medium completely from each well of the Bone Resorption Assay Plate and add 300μL/well of 5% sodium hypochlorite.

(2)    Incubate for five minutes at room temperature.

(3)    Wash the plate with tap water three times and with pure water once using a vat.

(4)    Dry the plate completely.

 

2. Observation of pits

(1)    Observe the pit formation in each well at 40x magnification. Pits will appear as individual or multiple clusters at the bottom of the well.

(2)    After focusing on the pits, turn off the light of the microscope.

(3)    Photograph pits in the well using a microscope with digital camera. The pits will be seen as black hole, and remaining calcium phosphate layer is as bright or white region.

(4)    Photograph five different regions in each well.

 

3. Measurement of pit area using Scion image software

(1)    Convert the all photo to the black and white image and enhance the contrast using Image software (Microsoft Office Picture Manager, Photoshop, etc).

(2)    Save the image as bitmaps. JPEG image cannot be analyzed by Scion Image Software.

(3)    Open an image of pits by Scion Image software.

(4)    Convert to a binary format image by using thresholding mode in order to distinguish between the pit area and background.

(5)    Measure pit area. Use “Show Results” to display the list of area measurement.

(6)    Measure all image under the same condition.

(7)    After finishing the measurement, “Export” and save the result.

Open the exported result file with an appropriate software and calculate the pit area in each well.

 

2011.6.15ver

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To be used for research only. DO NOT use for human gene therapy or clinical diagnosis.


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