I am trying to attach streptavidin-coated (actually Neutravidin from Molecular Probes) nanoparticles (90 nm, 100 nm, and 460 nm) to the surface of cultured 8E5 cells (human CD4+ T cells) via various primary biotinylated antibodies (CD3, CD4, CD14, CD45). However, I have noticed I get a lot of nonspecific binding of the NPs to the surfaces of the cells (confocal microscopy with these fluorescent NPs confirms this), even when I use a blocking solution (BlockAid, from Molecular Probes) on the NP before adding them to the cells.
I have confirmed that the 8E5 cells are CD14 negative via FITC CD14 Ab. I try to attach the NPs via CD14 as a negative control, expecting very few--if any--NP to attach. However, I get a lot of attachment!! Almost comparable to the positive test of attachment via CD45.
Below is a quick protocol:
1. Wash cell suspension 2x in PBS+1%BSA and finish with 100 uL volume.
2. Add biotinylated primary antibody (CD3, 4, 14, or 45). Only one type of Ab per tube of cells (no multiple staining). Incubate on ice for 30 min., with intermittent swishing. Should get rid of excess Ab.
3. Wash cells 2x in PBS+1%BSA and finish with final 100 uL volume.
4. Add NPs to cells and incubate on ice for 20 min. with intermittent swishing. Beforehand, NPs were placed in BlockAid solution and sonicated for 5 minutes.
5. Wash cells 3x in PBS+1%BSA and finish with 500 uL volume. Should get rid of extra NPs.
6. Evaluate cells using fluorescent microscope, confocal scope, or flow cytometry.
Has anyone else had this problem, or know what I am doing wrong?
I have been told about endogenous biotin in cells, however I think this is inside the cell (mitochondria). I am only worried about the surface of the cell. Could there still be endogenous biotin on the cell surface?