Leukocyte Alkaline Phosphatase Kit

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	PDL cells were fixed by immersion in a citrate-acetone-formaldehyde fixative solution for 5 min. The alkaline-dye mixture was prepared by mixing 1 mL Sodium Nitrite Solution and 1 mL of a fast red violet alkaline solution dissolved in 45 mL of distilled water and 1 mL of Naphtol AS-Bl alkaline solution. Surfaces were then placed in an alkaline dye mixture solution for 15 min protected from light followed by rinsing in deionized water.	All images were captured on a Wild Heerbrugg M400 ZOOM Makroskop (WILD HEERBRUGG, Heerbrugg, Switzerland) at the same magnification and light intensity and imported into Image J software (NIH, Bethesda, MD, USA). Image segmentation (Thresholding) was used to generate percent stained values for each field of view.

Protocol tips
PDL cells were fixed by immersion in a citrate-acetone-formaldehyde fixative solution for 5 min. The alkaline-dye mixture was prepared by mixing 1 mL Sodium Nitrite Solution and 1 mL of a fast red violet alkaline solution dissolved in 45 mL of distilled water and 1 mL of Naphtol AS-Bl alkaline solution. Surfaces were then placed in an alkaline dye mixture solution for 15 min protected from light followed by rinsing in deionized water.
Downstream tips
All images were captured on a Wild Heerbrugg M400 ZOOM Makroskop (WILD HEERBRUGG, Heerbrugg, Switzerland) at the same magnification and light intensity and imported into Image J software (NIH, Bethesda, MD, USA). Image segmentation (Thresholding) was used to generate percent stained values for each field of view.

Publication protocol

PDL cells were stimulated within each concentration of HA with and without ODM. At 7 days, cells were quantified for alkaline phosphatase expression utilizing a cell imaging system. Alkaline phosphatase activity was monitored using a Leukocyte alkaline phosphatase kit (procedure No. 86, Sigma) as previously described [33]. PDL cells were fixed by immersion in a citrate-acetone-formaldehyde fixative solution for 5 min. The alkaline-dye mixture was prepared by mixing 1 mL Sodium Nitrite Solution and 1 mL of a fast red violet alkaline solution dissolved in 45 mL of distilled water and 1 mL of Naphtol AS-Bl alkaline solution. Surfaces were then placed in an alkaline dye mixture solution for 15 min protected from light followed by rinsing in deionized water. All images were captured on a Wild Heerbrugg M400 ZOOM Makroskop (WILD HEERBRUGG, Heerbrugg, Switzerland) at the same magnification and light intensity and imported into Image J software (NIH, Bethesda, MD, USA). Image segmentation (Thresholding) was used to generate percent stained values for each field of view.

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