Alkaline phosphatase, osteopontin, and osteocalcin are expressed during the differentiation of bone marrow cells into mature osteoblasts and osteoblasts form calcium tubercle. Alizarin Red S, an anthraquinone derivative, has been used to identify Calcified nodule, because Calcium forms an Alizarin Red S-calcium complex, and the products turn (became) red. Calcified Nodule Staining kit (PMC-AK21-COS) contains Substrate-containing Buffer composed mainly of Alizarin Red S and Chromogenic Substrate, and provides a convenient system for staining calcified nodule with ease
|Substrate-containing Buffer (×100)||60 mL||4°C|
|Chromogenic Substrate||10 vials||4°C|
1. Remove culture medium. Wash each well with 1 mL of PBS. (× 3 times)
2. Remove PBS, and fix cells with 500 uL of methanol per well at room temperature for 30 minutes.
* Methanol should be cooled at -20 °C before use.
3. Remove methanol. Wash each well with 1 mL of distilled water. (× 3 times)
4. Add one vial of Chromogenic Substrate to 10 mL of Substrate-containing Buffer and mix, and then dispense 400 μL of the working solution to each well.
* This working solution should not be stored. Ready to use.
5. Incubate the plate at room temperature for 5 minutes
6. Wash each well with 1 mL of Substrate-containing Buffer until washing buffer become transparent.
Cells detected by Calcified Nodule Staining kit
|A: 3T3-E1 cells are cultured using Osteogenesis Culture for Osteogenesis Culture kit (Mouse) (Code No. PMC- OGCMO-COS) (35 mm dish)
B: Cells in Osteogenesis Culture Kit (Mouse) (PMC-OGC11-COS) (24well plate)
Calcified nodule Staining kit
To be used for research only. DO NOT use for human gene therapy or clinical diagnosis.