4 replies to this topic

[Henry Eub]

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.

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Edited by Henry Eub, 27 August 2009 - 12:26 PM.

[miss montana]

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:

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.

[CellSpecific.com]

You may want to use a different brighter fluor conjugate (e.g., PE-anti-CD14). Another is to use biotinylated anti-CD14 as your primary staining antibody and follow with streptavidin-FITC (or PE) to amplify your signals and increase separation.

[grayhair]

What cells are staining? Lymphocyes?
CD14 is monocytic marker and CD15 is a granulocytic marker.

[Clare]

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 28 August 2009 - 04:45 AM, 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.

Henry Eub, on Aug 27 2009, 11:35 AM, said:

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.