Hi All,
I want to do enzyme assays (SOD and Catalase) in chondrocytes from human articular cartilage. Please suggest me how many cells to be seeded in a petriplate or t75 flasks. I tried doing with a confluent petriplate I got very less concentration rather negligible amount of protein. What can be the cell count that is ideal to get good amount of protein?
Dear Saikrishna, You did not mention to what kind of enzyme assay. You need determine enzyme (SOD & CAT) amount? or Enzyme activity?
For measuring enzyme amount, you can use ELISA method for SOD and also for CAT.
in this situation you can prepare cell lysate with suitable lysis buffer and you need 10,000 cells averagely (10E4).
If you want determine enzyme activity, based on your intervention which induce or suppress enzyme expression you need 10,000 to 1000,000 cells. averagely you can determine enzyme activity with 100,000 cells (10E5).
best
mehdi
Thank you Sir!
I am actually following a protocol where they measure the SOD and the Catalase at 325 nm and 240 nm respectively in a UV spectrophotometer. And I want to know whether the 100,000 cells you mentioned is for seeding or it is the count at confluency? I am able to get 3x105 cells from a confluent petriplate ... is that count sufficient for making a cell lysate and extract protein for using in enzyme assays? or do we need more cells for getting enough amount of protein ...
I am using cell lysis buffer with the following ingredients -
In 0.05 M phosphate buffer
100 mM NaCl
1mM EDTA
2mM MgCl2
1mM PMSF
Try using a T150 if you don't have enough material for you assay. You can easily get 100,000 chondrocytes at 80% confluence.
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