Date: Thu Dec 20 2007 - 02:04:35 EST
Thanks to all who responded. Once again proof that Flowers are a caring, sharing community, just as I tell all the students attending our Flow Cytometry Courses. I have forwarded the replies to my colleague who will study them and try out your suggestions in the New Year. All the replies to date are posted below. Best wishes to all for 2008, on a glorious summer day (30+ oC) in Cape Town John Michie +27 21 9389 539 +27 21 933 8886 083 449 2225 On Dec 13, 2007, at 4:05 AM, Michie, John, Dr <jm5@sun.ac.za> wrote: A colleague has the following problem: After stimulation of PBMCs with PHA the cells become sticky. Trying to analyse on flow shows reduced numbers of cells compared to fresh. Cause of stickiness? Any help flowers? Reply 1. What is the cause I have never found out, but the phenomenon is experienced by many people. PHA is the worst stimulant if You want to use FACS for any analysis. Cells form clumps and it is quite difficult to get them all out of the well. I have managed to overcome some of the problem by incubating the cells after harvesting in RPMI 1640 medium containing 100 mg/ml N-acetylgalactosamine at 37 °C for 20 min, my volume for the incubation was 100 microliters per million cells. Cells are free from the clumps after this procedure and it is possible to count them in the haemocytometer, have never tried FACS after N-acetylgalactosamine - would imagine it can interfere with some surface proteins. Other stimulants like enterotoxin or concavalin-A do not cause this problem - PBMCs stay in nice suspention and FACS is no problem. anti-CD3 (+IL-2) can also be used but of course is more expensive. I would be grateful if You could let me know about other suggestions you recieve. Good luck Kate Dr Katarzyna Ruckemann-Dziurdzińska Katedra i Zakład Fizjopatologii Akademia Medyczna w Gdańsku ul. Dębinki 7 80-809 Gdańsk tel. +48 58 3491511 fax. +48 58 3491510 kruck@amg.gda.pl Reply 2. Probably lots of cell death. Try adding 20ug/ml DNaseI to the media & washes. bunny@cotleur.com Reply 3. DO you have 1mM EDTA in the staining buffers? SRW Susan.Watson@ucsf.edu Reply 4. PHA (phytohemagglutinin A) causes aggregation of red cells and also white blood cells to a considerable extent. PBMC is purified over Ficoll to primarily enrich mononuclear cells. We routinely culture PBMC with PHA and notice that a fraction (5 to 10%) will die by apoptosis within a day or two. However, if you want to analyze the cells by flow cytometry, it is easy to disrupt the clumps using a glass or disposable pasteur pipet. Pipet 3 or 4 times, collect the cells, centrifuge and resuspend in ca, Mg-free Dulbecco's PBS (Invitrogen) + 0.2% BSA to minimize clumping. Cells can be stained in this buffer (+ 0.01% sodium azide) on ice for 20 minutes, washed with the same buffer, fixed in 1% freshly prepared paraformaldehyde and analyzed without any problem. SJ S. Jayaraman, Ph.D. Associate Professor of Surgery University of Illinois at Chicago 909 South Wolcott Avenue-704E MSB-M/C 790 Chicago, IL 60612 Phone: 312-355-5133 Fax: 312-355-1497 anue2468@uic.edu Reply 5. I don't really stimulate my PBMCs with PHA, but I have observed that they get sticky and a lot of doublets/aggregates show up in the scatter gate. I reduced this problem by adding a final concentration of 2% fetal bovine serum and 0.53mM EDTA to the PBS. Also, keeping them cold/on ice/4C is important in reducing their stickiness. Hope this helps. Nidal Muvarak flowphilly@gmail.com Reply 6. PHA is phyto-haemo-agglutinin, therefore is the cause of the stickiness. You can try washing your cells after stimulation and resuspending them a couple of times, or adding EDTA as is normally done for intracellular whole blood staining protocols (see BD website for a list of protocols). I don't know if the EDTA will change some expression in your stimulated cells though. Regards Mara Mara Rocchi BVM&S, PhD Flow Cytometry Manager Moredun Research Institute Pentlands Science Park Bush Loan, Penicuik EH260PZ Scotland, UK Mara.Rocchi@moredun.ac.uk Reply 7. You probably need to add a chelator, such as EDTA to reduce the stickiness. It's the activation which causes it. I also use filter topped tubes to reduce the clumps running through my cytometer, but you do lose some cells this way. Betty-Anne bmcbey@uoguelph.ca Reply 8. PHA causes severe cells agglutination and it is difficult if not impossible to break up the aggregates. PHA M for has stronger agglutinating properties than PHA P, but the latter also agglutinates the cells. The aggregates tend to disperse after 48 h of incubation. Pokeweed Mitogen (Phytolacca) is another polyvalent mitogen - it does not induce agglutination but it activates B cells. Zbigniew Darzynkiewicz, M.D., Ph.D. Professor of Pathology and Medicine Director, Brander Cancer Research Institute New York Medical College BSB, Room 438 Valhalla, N.Y. 10595 www.darzynkiewicz.com/zbigniew/ <http://www.darzynkiewicz.com/zbigniew/> Z_DARZYNKIEWICZ@NYMC.EDU] Reply 9. PHA causes some cell death which releases DNA which causes cells to stick, presumably via a charge-charge interaction. A 5 minute incubation with 3500 Dornase units/ml of DNAse (in a Mg++ containing buffer!) will liberate them. DNAse details on page 6.24.4. [of attached pdf file] Calman cprussin@niaid.nih.gov Reply 10. It is a natural phenomenon that PHA stimulation of PBMC results in cluster formation, as phytohemagglutinin agglutinates blood cells (according to its name). Moreover, polyclonal cell activation results in massive upregulation of adhesion receptors, thus these cells stay together after division and will also exercise increased stickiness during sample processing. Nevertheless, we have routinely used mitogen stimulated cells in flow analysis, so as many others, especially since inception of division tracking dyes. Some points for handling stimulated cells: i) Careful pipetting while harvesting cells from culture plates is usually enough for disrupting the clusters. The effect can be controlled by microscopy or simply observing cells in wells in an inverted microscope. At the minimum, one should check the disrupting effect while establishing one's routine resuspending technique. In case of clinical diagnostic procedure, we specified the number of times for expelling the content of a well and pipette volume to standardize resuspension in the SOP. ii) Make sure the sample is resuspended before acquisition on a flow cytometer, as the cells may also clump during staining and washing/centrifugation. This can also be checked in a microscope, especially if the flow picture casts any doubts. iii) Doublet discrimination strategies have to be in place, so as exclusion of occasional "too large" events. Merry Holidays and Happy New Year to every one! Vladislav Rozenkov rozenkov@netscape.net
