What I do for lysis of heparin or EDTA, post antibody staining, is use "Whole Blood Lyse and Fix" by Gentrak....e-mail me for the address. First, antibody is added to 100 ul whole blood, then the lysing solution is added followed by the fixing solution, washed then I add 300-500 ul 2% paraformaldehyde. I read 1-2 days later. > > As I mentioned recently, I will also ask fairly basic > questions, as short version for people that are in a hurry, > the full story for interested people further down: > > - Has anyone experience with the lysis of heparinised blood > and scatter gating. How can I get rid of the thrombo > aggregates, what lysing solution is recommended? > > - Has anyone looked into the effect of formalin fixation on > the fluorescence of PE, or compared fluorescence intensities > of FITC and PE in different lysing solutions, perhaps NH4Cl > of distilled water. > > > > And here the long winded story: > Admittingly I am not an expert in clinical FCM, but want to > undertake a clinical study. I have consulted a number of > people and got the panel of appropriate antibodies. I > already asked some while ago about the use of transport > media for blood samples, but eventually we got transport > organized to get the samples 5 hour fresh. I would have > loved to get separate blood samples with heparin and EDTA, > but as 50ml heparinized blood is taken from the volunteers > for cell activity assays I eventually agreed that 3ml > heparinised blood would be fine to do both phagotest and > antibody staining (I remembered that heparin was a commonly > used anticoagulant in the early days), and this is were the > trouble starts. > I want to look at granulocytes (no gradients). I also would > like to avoid to spend the money on CD45 in every tube. As I > look at healthy volunteers, lymphocytes should not be rare > events, but a nice fat cluster. Fat it is indeed, covered in > a comet tail of activated thrombocytes. Initially I > compared a number of commercial lysing systems. They all > work fine - on EDTA blood, but can not handle Heparin. In > desperation and absence NH4CL lysing solution I used a > distilled water lyse neutralized with 10X PBS. This method > allows to distinguish the lymphos from the thrombo > aggregates already in a non-wash system but the lifetime of > the sample is limited (~2 hours). In addition my > fluorescence intensities are much higher, in FITC and PE > when compared to the commercial non-wash lysing solutions > (apart from the Dako Uti-Lyse). For CD14 this is a problem > as it goes out of scale and thus screws up the compensation. > Washing of the A.D.-lyse makes the gating easier as the > thrombos disappear. Whilst that is not the case with the > commercial reagents, at least the FITC fluorescence of those > samples recovers. Further investigation revealed that only > the Uti-lyse has a final pH that does not quench the FITC > fluorescence, but this does not explain the difference in > the PE fluorescence. > In the hope to keep my samples stable for longer I spun the > lysed samples and resuspendet them in PBS containing 0.4% > praformaldehyde (diluted to 289mOsmol, pH 7.4). Within two > hours my PE fluorescence comes down as well, but I have not > yet determined whether there is a final level or if it goes > down continuously. Perhaps the PFA only interferes with the > outer parts of the molecule. > If my observation is correct, it makes me wonder how to > compare signal intensities, or can't you do that with > fixative lysing solutions? Also the PE equivalents derived > from the Sperotech beads will thus be misleading as will be > the FCSC beads unless they have been lysed and fixed the > same way. > > I have searched the Howard 2nd edition (as someone else has > the 3rd), medline and cytometry, but without success. > Perhaps someone can give me a related reference or any > useful tips regarding the stabilization post lysis or any > minimum fixation time to wait for. > > > Probably having opened a can of nematodes I am looking > forward to lots of comments > > Gerhard Nebe-v.Caron > Unilever Research, Colworth, > Sharnbrook, Bedfordshire > GB - MK44 1LQ > Tel: +44(0)1234-222066 > FAX: +44(0)1234-222344 > gerhard.nebe-von-caron@unilever.com >
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