Fast semi－quantitative analysis of TRAP staining for osteoclast

HE You-hua; TONG Guo-jun; LI Ming-han; LI Wei; XU Fu-long; YANG De-hong

doi:10.3969/j.issn.1001-9448.2018.12.004

Abstract：Objective To select a suitable solvent for dissolving osteoclast tartrate resistant acid phosphatase stai -ning (the TRAP in situ staining); to explore the optimal wavelengths of light absorption ; and to compare with tartrate re-sistant acid phosphatase detection kit method , thus evaluate the stability of fast semi －quantitative analysis.Methods Seeding the bone marrow －derived macro－phages (BMMs) in 24 wells pretreated with plate collagen I.The cells were extracted from C57BL/6 mice, then divided into 4 groups, 10nmol/L PTH (1－34), 100 nmol/L PTH (1－34), 1 μmol PTH (1－34)/L and 10 μmol/L PTH (1－34) groups.The cells were incubated with PTH for 4 hours then replaced with a medium containing 10%fetal bovine serum for 44 hours, 4 h/44 h as a cycle.Eight days later, regular TRAP in situ staining was performed.Suitable solvents were screened for TRAP.Acetic acid was applied for final solved , with ultrason-ic grinding.Supernatant was collected for assessment of OD value with a suitable absorbance wavelength , and compared with tartrate resistant acid phosphatase kits.Results After TRAP in situ staining, acetic acid was chose as the optimized solvent.The 550 nm absorbance wavelength was ideal , when eliminating the interference of acetic acid.As the increasing of the concentration of PTH(1－34), the activity of osteoclasts increased , and the OD value increased as the same.The OD value of TRAP in situ staining solution and the OD value of tartrate resistant acid phosphatase detection kit method had significant correlation (r＝0.986, P＜0.014).Conclusion Dissolving the TRAP in situ staining with acetic acid , using 550nm absorbance wavelength can realize osteoclast rapid semi －quantitative analysis after dyeing.