monocyte isolation - (Jul/27/2002 )

hi, Can anyone suggest a method to isolate monocytes from whole blood?

Yeah, its quite easy with the ficoll-hypaque method. Dilute your buffy coat 1:2 with PBS. Prepare some 50 ml tubes with 25 ml ficoll (density 1.077 g/ml), then overlay the ficoll with an equal amount of the mixture from above. Centrifuge for 30 min with 700 x g without brake at 4 degress C. Soak up the white interphase between the plasma fraction and the ficoll fraction with a pipette and transfer it into a fresh tube. Wash twice with PBS. Then resuspend the cell pellet in medium and transfer the whole stuff to cell culture dishes (about 5 million PBMC per ml). The resulting PBMC suspension contains about 10% monocytes and 90% lymphocytes. Let the monocytes adhere for 1 h and wash away the non-adherent lymphocytes, so you get a nearly pure monocyte culture.

There's a commercial variation of the Ficoll Isopaque from Greiner we use for the Preparation of PBMC's from whole blood.

That System is called LeucoSep ?.

It includes 50ml Falcon Tubes containing a filter disk, so the seperation of MNC's and Ery's is near perfect.

Should be available at www.greiner-lab.com

Mike

You can also purify them through magnetic beads, stsrting from PBMC isolated as previously said. I recomend CD14 Microbeads from Miltenyi Biotec.

hi,
this paper gives nice idea about the technique " isolation of pure monocytes"
http://memorias.ioc.fiocruz.br/952/3879.html

hope it helps you.
gud luck

Hello,

Can I also use this method to purify blood cells from bone marrow specimen? Thank you.

Freshman using Ficoll-Hypaque

Dear payeli,

Thanks for your sharing,
I'm new to the isolation of monocytes.

From the article you recommended,
I've learned that the improved procedure comprises 2 major steps, one is Ficoll-Hypaque and the other is iso-osmotic Percoll gradient.

I'm much clear with the Ficoll-Hypaque procedure but not with the second step.

Can you or someone please advise the detailed procedure after removing the monocyte and lymphocyte layer from the centrifuged Ficoll-Hypaque gradient?

e.g. what's the volumn ratio when mixing the PBMC isolate into iso-osmotic Percoll solution?
After centrifugation, will there be several layers in the Percoll gradient?
Where will be the monocytes enriched layer that I should retreive?

Please provide some suggestions, thanks a lot!!

BR

Tim


[quote name='payeli' date='Mar 13 2006, 08:09 PM' post='43996']
hi,
this paper gives nice idea about the technique " isolation of pure monocytes"
http://memorias.ioc.fiocruz.br/952/3879.html

hope it helps you.
gud luck

Hi Tim,

this is some more detailed additional Info I recieved from Almeida (A Simple Method for Human Peripheral Blood Monocyte Isolation) on request, it might help you:


The monocytes forms a discrete ( not very well defined) layer over the gradient eventually with clumps.

Isolation of monocytes
Monocyte isolation was performed as previously described with slight modifications.
Briefly, a two step procedure with single gradients in each step was used. PBMC were isolated from buffy coats (obtained from normal blood donors) by centrifugation over a Ficoll-Hypaque (400g, 30 min, 25-350C). The PBMC were washed three times with PBS/Citrate (1.49 mM Na2H2PO4 ; 9.15 mM Na2HPO4 ; 139.97 mM NaCl, 13mM C6H5Na3O7 ; pH 7.2 ) (100g,15 min, 25-350C) to avoid platelet contamination. Around 108 PBMC were then incubated (15-30 min, 370C) in 15ml of a solution formed by nine parts of RPMI 1640 medium (Sigma) supplemented by 2mM L-glutamine,15mMHEPES, 10% human serum and 50μg/ml gentamcin and one part of trisodium citrate (C6H5Na3O7) 3,8%(w/v) in 50 ml conical polypropylene tubes with loosed lids in a CO2 incubator. This
suspension was then layered( very gently) on top of a 15ml Percoll gradient [1:1 (v:v),1visosmotic
Percoll :1v- PBS/Citrate] and centrifuged (400g, 20-30 min, 25-350C).
Eventually is useful to prepare a more dense gradient with 11 volumes of
isosmotic Percoll plus 9 parts volumes of PBS/citrate to lay first and then the
1:1 gradient. The cells may sediment between these gradients. Before use,
keep all solutions in water bath at 37° C . When preparing the salt solutions I
prefer autoclaving them instead of filtering to avoid salt loss. The isosmotic
Percoll used for preparing the mentioned gradients is done using 9 (volumes)
of Percoll (Pharmacia d=1.130) plus one volume of NaCL 1.5 M. Sometimes
the manufacturer´s Percoll has some variations in density, pay attention. The
Percoll could also be precipitated, being no useful in this case. Before using
the Percoll for preparing the gradients agitate it gently, and also the gradient
solutions before layering them. All the volumes have to be exactly matched.
At the end of last centrifugation collect also the 2 upper thirds of gradient.
Wash then as after the Ficoll. Here you could use centrifugal forces higher
then 100g, avoiding monocyte loss. Use a swing out rotor and polypropylene
tubes.

Nine parts RPMI 1640 medium
• 2mM L-glutamine,
• 15mMHEPES,
• 10% human serum
• 50μg/ml gentamcin and


one part of trisodium citrate (C6H5Na3O7
• 3,8%(w/v)



in 50 ml conical polypropylene tubes with loosed lids in a CO2 incubator.

Good luck,

Jonas

Are you planning downstream molecular or cellular studies for you isolated monocytes?
For molecular studies Dynabeads CD14 from Invitrogen can be used directly on whole blood or buffy coat. This is a really easy method.
If you want to do cellular studies, you don't want your cells to be attached to beads, so you should use a kit called Dynabeads Untouched Human Monocytes, which removes all the other cells. Isolated cells are bead and antibody free and have not been influenced by the isolation, which gives a very high confidence in further cellular studies. The untouched kit can only be used on PMBC though, so you'll need to prepare those first. I use Lymfoprep to prepare PBMC before isolation.
Roald