SHED (10 × 104 per 60 mm culture dish) were cultured under dentinogenic conditions with 0 μM bilirubin, 50 μM bilirubin (Merck), and 50 μM bilirubin plus 10 μM pamidronate (Tokyo Chemical Industry) according to previous studies [13 (link)]. Two and 4 weeks after the dentinogenic induction, the cultures were harvested for the examination of gene and protein expression and calcium deposition, respectively.
Pamidronate
Manufactured by Tokyo Chemical Industry
Sourced in Japan
Pamidronate is a laboratory reagent used in the analysis and research of various chemical compounds and materials. It is a white crystalline solid that is commonly used as a standard reference material in analytical techniques. Pamidronate's core function is to serve as a reference point for the identification and quantification of similar chemical substances.
Automatically generated - may contain errors
Lab products found in correlation
Bilirubin, by Merck Group (5 mentions)
Bilirubin, by Tokyo Chemical Industry (2 mentions)
Multiscan go, by Thermo Fisher Scientific (1 mentions)
Cell counting kit 8 (cck8), by Dojindo Laboratories (1 mentions)
Tnf α, by Thermo Fisher Scientific (1 mentions)
7 aad, by Thermo Fisher Scientific (1 mentions)
Facsverse, by BD (1 mentions)
6 protocols using pamidronate
1
Bilirubin and Pamidronate Effects on SHED Dentinogenesis
2
SHED Viability under Bilirubin and Pamidronate
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SHED (10 × 104/well) were seeded on 96-well multiplates and were incubated 24 h. Then, the cells were cultured with 0 μM bilirubin, 50 μM bilirubin (Merck), and 50 μM bilirubin (Merck) plus 10 μM pamidronate (Tokyo Chemical Industry) in the absence of serum for 3 days. Viability of the cells was assayed using a Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) according to the manufacturer’s instructions and was measured at OD 450 nm on a plate-reader Multiscan GO (Thermo Scientific, Walthan, MA).
3
Bilirubin and Pamidronate Effects on SHED
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SHED were seeded at 1 × 103 on 8-well chamber slides and were cultured for 24 h. The cells were incubated for 8 h under a serum-depleted condition and were subsequently stimulated with 0 μM bilirubin, 50 μM bilirubin (Merck), and 50 μM bilirubin (Merck) plus 10 μM pamidronate (Tokyo Chemical Industry) in the absence of serum for 3 days. As a control, SHED were treated with tumor necrosis factor alpha (TNFα; 10 nM, Peprotech, Rocky Hill, NJ). The cultures were fixed with 4% paraformaldehyde in PBS and analyzed by immunofluorescent microscopy.
4
Effect of Bilirubin and Pamidronate on SHED Bone Formation
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SHED were pretreated with 0 μM bilirubin, 50 μM bilirubin (Merck), and 50 μM bilirubin plus 10 μM pamidronate (Tokyo Chemical Industry) for 7 days. The cells (4 × 106) were mixed with hydroxyapatite tricalcium phosphate (40 mg, Zimmer Inc., Warsaw, IN) and were subcutaneously transplanted into 8- to 10-week-old female Balb/c nude/nude (Japan CLEA, Shizuoka, Japan) as described previously [2 (link)]. The implants were harvested 4 weeks after the transplantation. Frozen sections were treated for hematoxylin and eosin staining or immunofluorescent microscopy. The mineralized tissue area is represented as a percentage of the total area as described previously [2 (link)].
5
Bilirubin and Pamidronate Effects on Cultures
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Unconjugated bilirubin (Merk, Darmstadt, Germany) was diluted as according to the previous report [13 (link)]. Pamidronate (Tokyo Chemical Industry, Tokyo, Japan) was diluted in distilled phosphate buffered saline (PBS). In this study, we examined three group treated with 0 μM bilirubin, 50 μM bilirubin, and 50 μM bilirubin plus 10 μM Pamidronate. Control cultures were treated with the same amount of NaOH and albumin without bilirubin and/or PBS.
6
Bilirubin-Induced Apoptosis in SHED
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SHED were seeded at 10 × 103 and 100 × 103 cells per dish on 35-mm and 60-mm culture dishes, respectively, and were incubated for 24 h. Then, the cells were cultured with 0 μM bilirubin, 50 μM bilirubin (Merck), and 50 μM bilirubin (Merck) plus 10 μM pamidronate (Tokyo Chemical Industry) in the absence of serum for 3 days. For the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay, the cells were treated with the ApoTag Peroxidase In Situ Apoptosis Detection kit (Merck) according to the manufacturer’s instructions. The results are represented as a percentage of the numbers of the TUNEL-positive nuclei to the total ones with nucleated cells as described previously [13 (link)]. Moreover, the cells were stained with R-phycoerythrin-conjugated Annexin-V (eBioscience, San Diego, CA) and 7AAD (eBioscience) according to the manufacturer’s instructions and were analyzed with a FACS Verse flow cytometer (BD Bioscience, Franklin Lake, NJ).
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