In response to Howard Shapiro's comments about PHK26 The usefulness of PKH26 depends on your instrument setup. On the standard Facscan/FacSort (and FacsCaliber, I believe) filter setups, PKH26 will give you a very strong signal in both the FL2 and FL3 Channel. If you have a very, very bright flourochrome on your FL3 antibody, you may be able to run high enough compensation to remove the PHK26 signal from your FL3 PMT. I was only able to do this once using PerCP CD45 in conjunction with PKH26 labelled Human PBMC's. Seems to me like the compensation was something like 78% or greater to remove the PKH26 signal from the FL3 channel and see the remaining PerCP signal. For all practical purposes on the BD instruments mentioned above, PKH26 makes your instrument into a 2 flourescent channel instrument-- FL1 for FITC, and FL2 and FL3 for PKH26. The PKH26 signal will easily reach the 4th decade on these instruments in both FL2 and FL3. BUT as an added benefit, PKH26 is initially bright enough to be visible on a regular light microscope at high magnification. You can actually see little pink dots of the dye in the cell membrane. ALSO PKH26 is visible under flourescent microscopes for up to 42 days after implantation in spleen tissue in models like the SCID-HU-PBL mouse model using frozen sections. The standard setup for Phycoerythrin will work very nicely on most flourescent microscopes for PKH26. REMEMBER this dye is ALCOHOL soluble!!!! Do not submit tissue specimens for standard H and E staining and expect to see the PKH26 in the tissues. The dehydration process in H and E staining uses alcohol, which will remove the PKH26 from your sections. Labelling with PKH26 is not very complicated, but you do need to do a titration with the cells of interest to establish the proper concentration for labelling each cell line. A simple grid staining process and about 2 hours time will establish this for you. Once you have the concentration needed for the cells in question, I have labelled up to 5 x 10 to the ninth Human PBMC's in less that 2 hours for implantation into SCID mice. Unless you are really interested in tracking the doubling time for the cells I'd not recommend that you use PKH26. The labelling process involves some contact with ethanol, and you will change the permeability of your cell membranes as you do this. For example, immediately after staining with PKH26, all your cells may accept Trypan Blue and give you a very dim blue color as though they might be about to crump (die). In the labelling process you will also lose somewhere between 20 to 50% of your cell numbers due to centrifugations and plain old cell death. Some of this comes from having to put the cells into a system that is free of exogenous protein during the staining process. You will probably get a nice little snotty clumping of some cells that are dying. (You gene jockeys will know what I mean--its like a little snotty DNA clot) BUT--The PKH26 stained cells that survive are, however, quite viable, and based on studies with human PBMC's and CEM cells in tissue culture, have somewhat faster doubling times for the first few generations when compared with cells from the same donor that are not labelled with PKH26. Whether this is due to stimulation or increased permeability I do not know. Dennis Broud, Microbiologist DAPR,OTR, OPS, FDA HFD-910, MOD1, Room 3307 8301 Muirkirk Road Laurel, MD 20708 Phone (301) 594-5879 The comments above are the author's own, and do not in anyway reflect an official position by the US Food and Drug Administration or the United States Government.
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