John Parker wrote: > Hi Flowers, > > I am a relative neophyte to flow and I am having some problems developing an > assay to detect parvovirus (canine parvovirus infected cells). I have a > purified mouse mab against the capsid protein which I have directly > conjugated to Cy2. When I use this ab to stain infected cells on slides, I > see a very clean separation of infected from non-infected cells, almost all > the infected cell staining is in the nucleus and non-infected cells have no > or negligible background. I am using mv1Lu cells, a mink lung cell line. > However, when I trypsinize the cells from dishes and fix with 3% > paraformaldehyde, then permeabilize with 0.1% triton in PBSA , stain and do > flow. What I see is one broad peak which is shifted to the right on FL1 when > compared to a narrow peak of mock infected cells treated the same way and > stained with the same antibody. My interpretation of this is that I am not > fixing the antigen in the cells and that it subsequently leaks and coats the > surface of non-infected cells, hence the broad peak shifted to the right. My > questions are: > Is my interpretation reasonable, any other ideas? Any ideas on how I can > solve this problem or pin-point what is happening? > > Thanks in advance for your help > John John,First, a question . . . how does your PFA-Triton treatment compare with the protocol you use when looking at slides? Since you get good differential labeling in the latter, I would take clues from that protocol to develop your flow technique. This doesn't always work, however . . . you might have to dramatically alter your fix/perm technique to get this to work in flow. Now, assuming you've developed a protocol for revealing the antigen that 1) retains the antigen, and 2) preserves its antigenicity . . . then perhaps your problem lies in the need to clear non-targeting excess antibody. This may simply require additional washing, or may require that you supplement your wash buffer with a little Triton to assist in the clearing. Beyond that, you could look into 1) eliminating background due to excessive PFA (causes increased autofluorescence), or 2) look into enhancing the signal by modifying the emission filter. Keep in mind . . . a fluorescence scope will rarely have filter sets comparable to those in a flow, unless you specifically set it up that way. So, your flow cytometer may not be selecting the proper emission wavelength, thus eliminating some of the specific signal. MAK. -- Mark A. KuKuruga, Managing Director University of Michigan Core Flow Cytometry kukuruga@medmail.med.umich.edu
This archive was generated by hypermail 2b29 : Wed Apr 03 2002 - 11:52:57 EST