Osteoblast Culture kit V-1 - Cosmo Bio Co.,Ltd.

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Osteoblast Culture kit V-1

Catalog No.: PMC-OBC02-COS
Size: 1SET
Price: ¥114000
$1520
catalog info: Catalog 2012-p268
Storage: -20C & Liquid Nitrogen
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Purpose: For osteogenic capability
component: Osteoblasts (Frozen, 1~106 cells) x 1 vial
Culture Medium (500 mL) x 1 bottle (Cat. No. PMC-OBCM-COS)
Materials required but not provided
Variable volume pipettes
Culture flasks

Other:

 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.
[Derived from]
SD Rat Premature Cranial Bone
[Protocols]
1-1. Cultured with the 25 cm2 flask
1. Thaw the culture media in a 37C water bath withgentleshaking.
2. Quickly place osteoblast vial in a 37C 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 4C at 600 x g for 5 minutes.
4. Remove the supernatant, re-suspend cells in 10 ml of culture medium and centrifuge at 4C 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 suspensionto 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. Subcultureof 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 37C water bath priortouse.
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 solutionand thenimmediately remove trypsin/EDTA solution.
6. Incubate the flask in a 37C 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 4C at 600 x g for 5 minutes.
8. After removing the supernatant, re-suspend cells in culture medium and centrifuge for 5 minutes at 4C at 600 x g for 5minutes.
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 seedingdensity is 30,000 cells / cm2



 IMAGE1



Rat Osteoblast

 IMAGE2



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

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

Reference:

•  Hisa, I.,et al., J. Biol. Chem. 286, 9787-9796 (2011)
•  Itoh, T.,et al., J. Biol. Chem. 285, 27745-27752 (2010)
 
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