FLUORESCENT LABELLING OF MC3T3 CELL LINE BY 5-(AND-6)-CARBOXY-2', 7'-DICHLOROFLUORESCEIN DIACETATE, SUCCINIMIDYL ESTER MIXED

MC3T3 preosteoblast cell line의 5-(and-6)-carboxy-2',7'-dichlorofluorescein diacetate, succinimidyl ester mixed에 의한 fluorescent labelling

  • Kook, Min-Suk (Department of Oral & Maxillofacial Surgery, School of Dentistry, Dental Science Research Institute, Chonnam National University)
  • 국민석 (전남대학교 치의학전문대학원 구강악안면외과학교실)
  • Published : 2005.12.31

Abstract

Background. 5-(and-6)-carboxy-2',7'-dichlorofluorescein diacetate, succinimidyl ester mixed (CFSE) is the fluorescent labelling agent of living cells and used to trace the cells in vivo after transplatnation of various cells. The CFSE labelled cells can maintain fluorescence for up to 7 days after labelling. The MC3T3-E1 cell line (MC3T3) has been used for many studies about osteoblast, which is well known as a mouse preosteoblast. So the CFSE would be used to trace the transplanted MC3T3. However there are few reports about CFSE labelling of MC3T3. This study is aimed to know about adequate concenturation and incubation time of CFSE to MC3T3. Materials and methods. The MC3T3 was incubated in a humidified atmosphere of 95% air with 5% $CO_2$ at $37^{\circ}C$ using ${\alpha}$-minimal essential medium (${alpha}$-MEM) containing10% FBS and gentamycin. Ten mM CFSE solution in dimethylsulphoxide (DMSO: 1%) was diluted with phosphate buffered saline (PBS) and final concentration of culture medium was, respectively, 5, 10, 15, 20, 25 and 30 ${{\mu}M$. Then the MC3T3 was incubated with CFSE in a humidified atmosphere of 95% air with 5% $CO_2$ at $37^{\circ}C$ for 5, 10, 15, 20, 25, 30, 35, 40 and 45 minutes in each concentration. The fluorescence of CFSE labelled cells was analysed with a inverted fluorescence microscope. The duration of cell labelling was also studied. Trypan blue dye exclusion test was done for cell viability. Results. For concentration between 5 and 10 ${\mu}M$, CFSE did not significantly label the MC3T3 in vitro. The destruction of MC3T3 was observed at the concentration of 20 ${\mu}M$. In the concentration of 15 ${\mu}M$, the best labelling was obtained at an incubation period between 15 and 30 minutes. The MC3T3 labelled with an incubation period of 15 minutes at 15 ${\mu}M$ was still fluorescent 7 days after CFSE labelling. The mean cell viability was 95.93%. Conclusion. These results suggests an incubation period of 15 minutes at 15 ${\mu}M$ of CFSE provides best labelling of MC3T3 in vitro.

Keywords

References

  1. Oehen S, Brduscha-Riem K, Oxenius A, Odermatt B: A simple method for evaluating the rejection of grafted spleen cells by flow cytometry and tracing adoptively transferred cells by light microscopy. J Immunol Methods 1997;207(1):33-42 https://doi.org/10.1016/S0022-1759(97)00089-6
  2. Garton HJ, Schoenwolf GC: Improving the efficacy of fluorescent labelling for histological tracking of cells in early mammalian and avian embryos. Anat Rec 1996;244(1):112-117 https://doi.org/10.1002/(SICI)1097-0185(199601)244:1<112::AID-AR11>3.0.CO;2-S
  3. Paramore CG, Turner DA, Madison RD: Fluorescent labelling of dissociated fetal cell culture for tissue culture. J Neurosci Methods 1992;44(1):7-17 https://doi.org/10.1016/0165-0270(92)90108-P
  4. Vroemen J: Transplantation of isolated hepatocytes into the pancreas.Eur Surg Res 1988;20:1-11
  5. Weston SA: New fluorescent dyes for lymphocyte migration studies. Analysis by flow cytometry and fluorescent microscopy. J Immunol Methods 1990;133:87-96 https://doi.org/10.1016/0022-1759(90)90322-M
  6. Simain-Sato F, Lahmouzi J, Heinen E, Defresne MP, De Pauw-Gillet MC, Grisar Th, Legros JJ, Legrand R: Graft of autologous fibroblasts in gingival tissue in vivo after culture in vitro preliminary study on rats. J Periodont Res 1999;34:323-328 https://doi.org/10.1111/j.1600-0765.1999.tb02260.x
  7. Rey C, Kim HM, Gerstenfeld L, Glimcher MJ: Characterization of the apatite crystals of bone and their maturation in osteoblast cell culture: comparison with native bone crystals. Connect Tissue Res 1996;35:343-349 https://doi.org/10.3109/03008209609029210
  8. Bhargava U, Bar-Lev M, Bellows CG, Aubin JE: Ultrastructural analysis of bone nodules formed in vitro by isolated fetal rat calvarial cells. Bone 1988;9(3):155-163 https://doi.org/10.1016/8756-3282(88)90005-1
  9. Arceo N, Sauk JJ, Moehring J, Foster RA, Somerman MJ: Human periodontal cells initiate mineral-like nodules in vitro. J Periodontol 1991;62:499-503 https://doi.org/10.1902/jop.1991.62.8.499
  10. Casser-Bette M, Murray AB, Closs EI, Erfle V, Schmidt J: Bone formation by osteoblast-like cells in a three-dimensional cell culture. Calcif Tissue Int 1990;46(1):46-56 https://doi.org/10.1007/BF02555824
  11. Nolan PC, Nicholas RM, Mulholland BJ, Mollan RA, Wilson DJ: Culture of human osteoblasts on demineralised human bone. Possible means of graft enhancement. J Bone Joint Surg Br 1992;74(2):284-286
  12. Ishaug SL, Crane GM, Miller MJ, Yasko AW, Yaszemski MJ, Mikos AG: Bone formation by three-dimensional stromal osteoblast culture in biodegradable polymer scaffolds. J Biomed Mater Res 1997;36(1):17-28 https://doi.org/10.1002/(SICI)1097-4636(199707)36:1<17::AID-JBM3>3.0.CO;2-O
  13. Roth JA, Kim BG, Lin WL, Cho MI: Melatonin promotes osteoblast differentiation and bone formation. J Biol Chem 1999;274(31): 22041-22047 https://doi.org/10.1074/jbc.274.31.22041
  14. Dean DD, Schwartz Z, Bonewald L, Muniz OE, Morales S, Gomez R, Brooks BP, Qiao M, Howell DS, Boyan BD: Matrix vesicles produced by osteoblast-like cells in culture become significantly enriched in proteoglycan-degrading metalloproteinases after addition of $\beta$-glycerophosphate and ascorbic acid. Calcif Tissue Int 1994;54:399-408 https://doi.org/10.1007/BF00305527
  15. Dean DD, Schwartz Z, Bonewald L, Muniz OE, Morales S, Gomez R, Brooks BP, Qiao M, Howell DS, Boyan BD: Matrix vesicles produced by osteoblast-like cells in culture become significantly enriched in proteoglycan-degrading metalloproteinases after addition of $\beta$-glycerophosphate and ascorbic acid. Calcif Tissue Int 1994;54:399-408 https://doi.org/10.1007/BF00305527
  16. Roth JA, Kim BG, Lin WL, Cho MI: Melatonin promotes osteoblast differentiation and bone formation. J Biologic Chem 1999;274(31): 22041-22047 https://doi.org/10.1074/jbc.274.31.22041
  17. Ilana Y, Idit S, Rachel J, David L, Dan C, Michael Y: Fluoxetine and amitriptyline inhibit nitric oxide, prostaglandin E2, and hyaluronic acid production in human synobial cells and synobial tissue cultures. Arthiritis Rheum 1999;42(12):2561-2568 https://doi.org/10.1002/1529-0131(199912)42:12<2561::AID-ANR8>3.0.CO;2-U
  18. Partovi M, Sadeghein A, Azizi E, Ostad SN: Mitogenic effect of Ldopa on human periodontal ligament fibroblast cells. J Endodont 2002;28(3):193-196 https://doi.org/10.1097/00004770-200203000-00012
  19. Yasufumi I, Katsumi E, Kiyoshi M, Yojiro K, Toshiaki T, Takehiko K, Kuniko A, Takahiko A, Hideki N, Shigenobu N: Apoptosis induction in synobial fibroblasts by ceramide: in vitro and in vivo effects. J Lab Clin Med 1998;131(5):410-416 https://doi.org/10.1016/S0022-2143(98)90141-X
  20. Karrer FM, Reitz BL, Hao L, Lafferty KJ: Fluorescein labelling of murine hepatocytes for identification after intrahepatic transplantation. Transplant Proc 1992;24(6):2820-2821