Flow Cytometry abreview for Anti-HA tag antibody (Biotin)

Flow Cytometry

Human Cell (A3.01 human CD4+ T cell line engineered to express)

No

Gated on major population by FSC/SSC (i.e. excluding dead cells/debris)

A3.01 human CD4+ T cell line engineered to express

Cell harvesting/tissue preparation method: Suspension cells
Sample buffer: RPMI 1640/10% FBS

Formaldehyde

30 minute(s) · Temperature: 4°C · Diluent: RPMI 1640/10% FBS

1/500

1/500

Non-Abcam antibody was used: Invitrogen #T2767
Host species: Goat
Clonality: Polyclonal
Conjugation: Texas Red ®

The cells are A3.01 CEM-derived CD4+ T cells, transduced with a lentivirus for stable expression of a surface protein which contains HA tag in the extracellular region, and selected using Puromycin for several weeks, followed by anti-HA immunomagnetic enrichment (resulting in >95% cells expressing the protein on the surface, as determined by co-labeling for another epitope in the same protein). Cells were washed and surface-stained on ice for 30 min with RPMI/10% FBS containing the indicated antibody at the indicated dilution, then washed with PBS, resuspended in PBS/5 mM EDTA, and fixed with an equal volume of PBS/8% PFA (final 4% PFA) for 10 min @ RT. Cells were then washed with PBS, stained with goat anti-rabbit Texas Red secondary Ab or Texas Red-conjugated streptavidin (SA-T-Red), each at 1:500 dilution in PBS/1% BSA for 30 min @ RT, washed, and resuspended in PBS before flow cytometry analysis (MACSQuant VYB, 561 nm excitation, Y2/dsRed channel). 50,000 cells per tube were run, and cells were gated by FSC/SSC to exclude dead cells and debris. ab9110 is the unconjugated version of ab26228. Both are rabbit polyclonals at 1 mg/ml stock. Not shown: I also tried SA-TexasRed at higher concentrations, but the signal in HA-negative cells increased as much as that in HA-positive cells. Not sure why the efficiency of streptavidin labeling is so poor, it could be the antibody is not fully biotinylated, or that my streptavidin is degraded, but I could not tell either way.