Platelet Contamination during PBMC Enrichment - (Aug/25/2011 )
Hello there,
I have been working for a long time with Biocoll for PBMC Isolation. My goal is to isolate highly purified Monocytes from various patients. I have been extracting PBMCs via Ficoll and then always made a positive monocyte isolation with microbeads. Recently our lab changed the method to a negative monocyte isolation kit which does not contain any anti platelet antibodies. Now I'm faced with a large contamination of platelets which i do not get rid off.
Here's my protocol:
1)collect 30 ml citrate blood
2)Dilution of 8ml blood with 27ml PBS withou Ca,Mg, 2mM EDTA (Buffer is stored at 4°C, Ficoll is stored at RT)
3)Layer solution over 15ml Biocoll
4)400g 35min without breaks at RT
5) Discard supernatant
6)Carefully aspirate Interphase
7) Dilution with PBS EDTA 2mM up to 50ml
8) 300g x 10min at RT
9)200g 10min
10) 200g 10min
11) Count cells -> Monocyte isolation via microbeads
My Platelet content is up to 30-40%, Lymphos 40%, Monocytes 10% after Biocoll
Does anybody have any additional strategy to avoid platelet contamination? I transport my samples on ice from the clinics. Today I tried everything at RT (even PBS) and also had large contamination with platelets.
I would be very grateful if somebody could help me
Alex
P.S Another problem I'm faced with is clotted red particels in the interphase which do not sediment with erythrocytes but which dissolve normally in PBS after aspiration of the interface and contaminate my sample too. This happened at several patients samples (10% maybe).
Hi Alexander,
Please see attached protocol.
In RD when we developed our kits for isolation of Untouched cells they used this protocol all the time to reduce the number of platelets to a minimum.
As we say "Crap cells in is crap cells out"!
Untouched cells is really the way to go to make sure you have as "naive" cells as possible, but this procedure will also reduce the purity.
To get as high purity as possible, the positive isolation road is the key.
I saw that you isolate you cells from whole blood.
If you want to know more on how to use one product for isolation of highly pure monocytes directly from whole blood followed by with removal of beads (Dynabeads) after isolation, let me know.
Or else good look with the PBMC protocol.
Regards Berit M Reed
Product Manger for Cell Separation, Dynabeads (Life Technologies)
I can be reached at :
Hi Berit,
thanks a lot for your recommendations, I'll try your protocol next week an will let you know about the results!
Regards Alexander
Hi Alexander
My name is Ketil Winther Pedersen and I am running the technical support for Dynabeads products located at the production facility in Oslo, Norway.
Please contact me at any time for technical questions regarding Dynabeads.
kind regards
ketil
*********************************
Ketil Winther Pedersen, Ph.D
Senior Technical Application Specialist,
Technical support for Dynabeads products
Life Technologies AS
Tel: +47 22 06 11 10 (direct)
Fax: +47 22 51 95 05
eurotech@lifetech.com
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