PHA stimulation of PBMCs results in sticky cells - The replies

rozenkov@netscape.net rozenkov at netscape.net
Wed Jan 2 22:29:14 EST 2008


So, the attached pdf is a part of selling campaign?


-----Original Message-----
From: Michie, John, Dr <jm5 at sun.ac.za> <jm5 at sun.ac.za>
To: cyto-inbox
Sent: Wed, 2 Jan 2008 8:01 pm
Subject: RE: PHA stimulation of PBMCs results in sticky cells - The replies



Thanks to all who responded. Once again proof that Flowers area caring, sharing 
ommunity, just as I tell all the students attending our Flow Cytometry Courses. 
 have forwarded the replies to my colleague who will study them and try out 
our suggestions.
All the replies to date are posted below and a pdf file sent by Calman Prussin 
Detection of Intracellular Cytokines by Flow Cytometry) is attached.
 
Best wishes to all for 2008, on a glorious Summer day 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 at sun.ac.za> wrote:
A colleague has the following problem:
After stimulation of PBMCs with PHA the cells become sticky. Trying to analyse 
n 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 
any people. PHA is the worst stimulant if You want to use FACS for any 
nalysis. Cells form clumps and it is quite difficult to get them all out of the 
ell. I have managed to overcome some of the problem by incubating the cells 
fter harvesting in RPMI 1640 medium containing 100 mg/ml N-acetylgalactosamine 
t 37 °C for 20 min, my volume for the incubation was 100 microliters per 
illion cells. Cells are free from the clumps after this procedure and it is 
ossible to count them in the haemocytometer, have never tried FACS after 
-acetylgalactosamine - would imagine it can interfere with some surface 
roteins. Other stimulants like enterotoxin or concavalin-A do not cause this 
roblem - PBMCs stay in nice suspention and FACS is no problem. anti-CD3 (+IL-2) 
an also be used but of course is more expensive. I would be grateful if You 
ould 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 at amg.gda.pl
 
Reply 2.
Probably lots of cell death. Try adding 20ug/ml DNaseI to the media & washes. 
bunny at cotleur.com
 
Reply 3.
DO you have 1mM EDTA in the staining buffers?
SRW
Susan.Watson at ucsf.edu
 
Reply 4.
PHA (phytohemagglutinin A) causes aggregation of red cells and also white blood 
ells to a considerable extent. PBMC is purified over Ficoll to primarily enrich 
ononuclear 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 
nalyze the cells by flow cytometry, it is easy to disrupt the clumps using a 
lass or disposable pasteur pipet. Pipet 3 or 4 times, collect the cells, 
entrifuge and resuspend in ca, Mg-free Dulbecco's PBS (Invitrogen) + 0.2% BSA 
o minimize clumping. Cells can be stained in this buffer (+ 0.01% sodium azide) 
n ice for 20 minutes, washed with the same buffer, fixed in 1% freshly prepared 
araformaldehyde 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 at uic.edu
 
Reply 5.
I don't really stimulate my PBMCs with PHA, but I have observed that they get 
ticky and a lot of doublets/aggregates show up in the scatter gate. I reduced 
his problem by adding a final concentration of 2% fetal bovine serum and 0.53mM 
DTA to the PBS. Also, keeping them cold/on ice/4C is important in reducing 
heir stickiness. Hope this helps.
Nidal Muvarak
flowphilly at 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 
f times, or adding EDTA as is normally done for intracellular whole blood 
taining protocols (see BD website for a list of protocols). I don't know if the 
DTA 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 at 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 at uoguelph.ca
 
Reply 8.
PHA causes severe cells agglutination and it is difficult if not impossible to 
reak up the aggregates. PHA M for has stronger agglutinating properties than 
HA P, but the latter also agglutinates the cells. The aggregates tend to 
isperse after 48 h of incubation. Pokeweed Mitogen (Phytolacca) is another 
olyvalent mitogen - it does not induce agglutination but it activates B cells. 
Zbigniew Darzynkiewicz, M.D., Ph.D. 
rofessor of Pathology and Medicine 
irector, Brander Cancer Research Institute 
ew York Medical College 
SB, Room 438 
alhalla, N.Y. 10595
ww.darzynkiewicz.com/zbigniew/ <http://www.darzynkiewicz.com/zbigniew/> 
_DARZYNKIEWICZ at NYMC.EDU]
Reply 9.
HA causes some cell death which releases DNA which causes cells to stick, 
resumably via a charge-charge interaction. A 5 minute incubation with 3500 
ornase units/ml of DNAse (in a Mg++ containing buffer!) will liberate them.
NAse details on page 6.24.4. [of attached pdf file]
alman
prussin at niaid.nih.gov
Reply 10.
t is a natural phenomenon that PHA stimulation of PBMC results in cluster 
ormation, as phytohemagglutinin agglutinates blood cells (according to its 
ame). Moreover, polyclonal cell activation results in massive upregulation of 
dhesion receptors, thus these cells stay together after division and will also 
xercise increased stickiness during sample processing. Nevertheless, we have 
outinely used mitogen stimulated cells in flow analysis, so as many others, 
specially since inception of division tracking dyes. Some points for handling 
timulated cells:
i) Careful pipetting while harvesting cells from culture plates is usually 
nough for disrupting the clusters. The effect can be controlled by microscopy 
r simply observing cells in wells in an inverted microscope. At the minimum, 
ne should check the disrupting effect while establishing one's routine 
esuspending technique. In case of clinical diagnostic procedure, we specified 
he number of times for expelling the content of a well and pipette volume to 
tandardize resuspension in the SOP.
ii) Make sure the sample is resuspended before acquisition on a flow cytometer, 
s the cells may also clump during staining and washing/centrifugation. This can 
lso be checked in a microscope, especially if the flow picture casts any 
oubts.
 
iii) Doublet discrimination strategies have to be in place, so as exclusion of 
ccasional "too large" events.
 
Merry Holidays and Happy New Year to every one!
 
Vladislav Rozenkov
rozenkov at netscape.net
 



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