I am trying to modify a mutation assay I have had success with in other suspension cell lines - PIG-A and have encountered some difficulties that I am hoping to troubleshoot with you all. PIG-A is a transmembrane (GPI) anchor, which functions to tether a variety of cell surface molecules to the cell surface. A null mutation in the PIGA gene results in the loss of this transmembrane anchor protein, and also the loss of all of these tethered cell surface receptors. Hence PIGA mutants can be detected by flow cytometry by looking for the absence of two or more of these cell surface receptors (to ensure that mutations in the genes of the cell surface receptors alone are not counted). I have had previous success with this assay in a lymphoblastoid cell line and generated some excellent mutation data which is about to be published. I am trying to adapt the assay for a myeloid cell line (HL-60), but am encountering problems.

The two GPI-anchored cell surface receptors I am looking for are CD55 and CD59, both of which are highly expressed on HL-60 and show significant binding with fluorochrome-conjugated antibodies.

As well as this, I am concurrently using an antibody to bind CD45 as a positive control and 7-AAD to exclude dead cells.

All antibodies show excellent binding, but even in 500,000 cells, I am not picking up a single one which appears to be a PIGA mutant (CD55 and CD59 negative). I have pre-treated the cells with an Fc Blocking agent to prevent non-specific binding of the antibodies and have checked the isotype controls to make sure there is no non-specific binding. I have also treated cells with radiation at a dose which I know should cause significant mutation as a positive control - and cannot see any PIGA mutations here either (8 days post treatment, which initially caused around 70% cell death). I would absolutely expect to see at least 5-10 PIGA mutant cells in 500,000 events, and far more in the radiation treated cells.

A few things I have considered:

I am using too much antibody and it is nonspecifically binding to everything causing false positives (I shall titrate it this afternoon and try with less antibody).

My compensation for the assay is incorrect and I am not seeing negative cells due to leakage from other channels (NB: All cells I use in the assay are EGFP+)

PIGA mutation is fatal in HL-60 but not in the previous lymphoblastoid cell line I have used.

HL-60 have a very low basal PIGA mutation rate and I have not left it long enough after the radiation treatment to allow the cells to fix the PIGA mutations and lose cell surface expression of CD55 and CD59.

Please note in attachment (gating strategy for PIGA) - CD19 is the control marker used in the lymphoblastoid cell line and is now CD45 in HL-60.

I realise that this is a quick summary of a not incredibly simple assay, which many will be unfamiliar with, so please do ask for more information/clarification if you think you might be able to help, i'll try to respond quickly.

Many thanks for any help - I'm at my wits end with this assay - there is no theoretical reason why it shouldn't work, but I am definitely not picking up any mutants at all.

Additionally - if anyone is interested in the assay - there has been a publication about it here (with different cell types to that I am using) http://www.ncbi.nlm.nih.gov/pubmed/20034593