Flow cytometry CD14 staining very dim - Bone marrow cells CD14 staining for flow cytometry (Aug/27/2009 )
I did CD14 staining on fresh Bone marrow cells for flow cytometry, I found it was very dim. neg and positive don't seperate very well, see the piture.
The antibody is FITC conjunct from ebioscience. I use 2ug for 100ul 1 x 10E6 cells, 10% rat serum blocking for 20 mins and then incubate with antibody for 30 mins at room temperature.
ANybody has any suggestions for the staining?
Thank you.
Is your compensation OK? It can be difficult to distingusih between antibodies in FITC and PE, especially when one of them is dim if the compensation is off. Your staining protocol sounds fine, although I usually incubate for 30 minutes on ice or in the fridge.
Henry Eub on Aug 27 2009, 11:35 AM said:
The antibody is FITC conjunct from ebioscience. I use 2ug for 100ul 1 x 10E6 cells, 10% rat serum blocking for 20 mins and then incubate with antibody for 30 mins at room temperature.
ANybody has any suggestions for the staining?
Thank you.
What cells are staining? Lymphocyes?
CD14 is monocytic marker and CD15 is a granulocytic marker.
Yup - I agree with Miss Montana - your compensation may not be ok. Can you post us your FSC/SSC and dead cell stains showing us your gates?
Clare
miss montana on Fri Aug 28 12:45:43 2009 said:
Is your compensation OK? It can be difficult to distingusih between antibodies in FITC and PE, especially when one of them is dim if the compensation is off. Your staining protocol sounds fine, although I usually incubate for 30 minutes on ice or in the fridge.
I did CD14 staining on fresh Bone marrow cells for flow cytometry, I found it was very dim. neg and positive don't seperate very well, see the piture.
The antibody is FITC conjunct from ebioscience. I use 2ug for 100ul 1 x 10E6 cells, 10% rat serum blocking for 20 mins and then incubate with antibody for 30 mins at room temperature.
ANybody has any suggestions for the staining?
Thank you.