Useful for evaluate osteogenic capability!

Osteoblast Culture Kit


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Background

Bone metabolism is composed of balanced bone formation of osteoblasts and bone resorption of osteoclasts.@Osteoblast Culture Kit (Rat) (PMC-OBC02-COS) contains frozen osteoblasts isolated from rat calvariae and culture medium. Osteoblast Culture Kit (Rat) (PMC-OBC02-COS) is a useful to study osteoblast and osteogenesis.

Rat Osteoblast

Staining with Alkaline Phosphatase Staining Kit (Cat#PMC-AK20-COS) of Rat Osteoblast

Staining with Calcified nodule Staining kit (Cat#PMC-AK21-cos) of Rat Osteoblast in confluent culture (12-well plate)

Kit component

Product Name Size Quantity Storage Conditions Shelf Life
Osteoblast, Rat 1 x 106 cells/vial 1 Liquid Nitrogen 1 year
Culture Medium
(Cat. No. PMC-OBCM-COS)
500 ml 1 -20°C 6 months

Protocols

<1-1. Cultured with the 25 cm2 flask> 

1. Thaw the culture media in a 37°C water bath with gentle shaking.

2. Quickly place osteoblast vial in a 37°C water bath until the contents are thawed.

3. Transfer thawed cells into a 15 ml centrifuge tube containing 10 ml of culture medium and centrifuge for 5 minutes at 4°C at 600 x g for 5 minutes.

4. Remove the supernatant, re-suspend cells in 10 ml of culture medium and centrifuge at 4°C at 600 x g for 5 minutes.

5. Remove the supernatant, and re-suspend the cell pellet in approximately 5 ml of culture medium.

6. Transfer the cell suspension to 25 cm2 flask and incubate the flask at 37oC under 5% CO2 and 100%@@humidity.

7. The next day, change the medium.

* Approximately 2-3 days of culture, cells become confluent. For subculture, please refer to the protocol below. Subculture of the cells can be performed up to passage 2. 

 

<1-2. Subculture >

1. Subculture the cells when they are confluent.

2. Prepare sterile washing buffer (Hank’s BSS or PBS(-)), and trypsin/EDTA solution. Warm washing buffer in a 37°C water bath prior to use.

3. Rinse the cells with 5 ml of washing buffer twice.

4. Remove washing buffer and then add 3 ml of trypsin/EDTA solution into flask (25 cm2 flask).

5. Gently rock the flask to make sure that the cells are covered by trypsin/EDTA solution and then immediately remove trypsin/EDTA solution.

6. Incubate the flask in a 37°C incubator until cells are completely rounded up (monitored with inverted microscope). Approximately it takes 2 to 3 minutes.

7. Add culture medium to the flask and transfer detached cells to centrifuge tube, and then centrifuge the centrifuge tube at 4°C at 600 x g for 5 minutes.

8. After removing the supernatant, re-suspend cells in culture medium and centrifuge for 5 minutes at 4°C at 600 x g for 5 minutes.

9. Remove the supernatant, and re-suspend cells in culture medium. Count cells and plate cells in a new plate or flask (Adjust cell density to the desired experiment).

* Approximately 2-3 days of culture, cells become confluent when seeding density is 30,000 cells/ cm2

References

> Hisa, I., Inoue, Y., Hendy, G. N., Canaff, L., Kitazawa, R., Kitazawa, S., Komori, T., Sugimoto, T., Seino, S., Kaji, H. Parathyroid Hormone-responsive Smad3-related Factor, Tmem119, Promotes Osteoblast Differentiation and Interacts with The Bone Morphogenetic Protein-Runx2 Pathway. J. Biol. Chem. 286, 9787-9796 (2011)

> Itoh, T., Takeda, S., Akao, Y. MicroRNA-208 Modulates BMP-2-stimulated Mouse Preosteoblast Differentiation by Directly Targeting V-ets Erythroblastosis Virus E26 Oncogene Homolog 1. J. Biol. Chem. 285, 27745-27752 (2010)

Product List

Product Name Cat# Quantity Price

Osteoblast Culture kit V-1 DataSheet ,

PMC-OBC02-COS

1 SET ¥114,000
$1520

Osteoblast Culture Medium DataSheet ,

PMC-OBCM-COS

500 ML ¥31,200
$416

To be used for research only. DO NOT use for human gene therapy or clinical diagnosis.


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