Thanks everyone for Your help, I appreciate it! I'll try to isolate PBMC from whole blood like you say.

the required protocol is 1. take equal amount of blood and ficol put for the incubation at 45 degree for one hour, you will have two clear layer, take the upper layer care fully with the normal saline and go for the centrifuge for 15 min at 5000 g this time a white ring will be present in the middle (there will be three layers) this ring will actually cantain the PBMC, take the same with the help of micropippate and centrifuge count and may be processed for any sort of treatment. Hope for the best.

hiii Oskan Tasinov

protocal

collect 10 milliliteries venous blood in heparininzed blood collection tubes.

sample must be processed within 4-6 h of collection.

centrifuge the collected samples at 2000rpm at 20c for 10 min.

aspirate the plasma into sterile cryo-vials and store at -70c.

Dilute the packed blood cell 1:1 in sterial RPMI media (in 50ml tubes).

carefully overlaid the suspension onto Ficoll- Hypaque layer.

centrifuge at 1400rpm for 25 min without brake.

carefully aspirate the monnuclear dense ring, at the RPMI- 1640plasma -hypaque interface, into fresh sterile 15ml centrifuge tubes.

wash the collected cells 2 times in 10ml RPMI- 1640.

centrifuge at 1200rpm for 10minutes after each wash.

Suspend cells pellet in 2ml of RPMI 1640 MEDIA enriched with 10% heat- inactivated fetal calf serum , complete culture media.

hope to be best and benefit

Histopaq (HIGH MEDIA) you can use for your work.

Take 3 ml histopaq and add 2 mall blood or you can use in 1;1 ratio the centrifuge it for 15 minutes at 5000 rpm.

you will get erythrocytes and leukocytes.

A little "education" here. A "buffy coat" is so called because when blood is centrifuged the dense red cells (and granular polymorphonuclear leukocytes) sink to the bottom. The lighter mononuclear leukocytes form a layer on top of the red cells which is pale cream or "buff" in colour, hence "buffy coat", which is below the plasma layer. The buffy coat may contain some platelets depending on how hard and/or long the blood was spun for and the remaining platelets will be in the plasma. In 1968 Boyum described a method known as buoyant-density centrifugation where the blood is layered over a medium of the correct density (1.077 for human) to allow the red cells and granulocytes to sink and the lighter mononuclear leukocytes to float when the blood is spun in a centrifuge. A number of companies sell this reagent including GE Healthcare, Sigma, Axis-Shield - and all have good method descriptions on their websites and product inserts. As well as having a density component the reagent is usually mixed with a dextran or starch polysaccharide to help red cells agglutinate and remove them more efficiently. It does not seem necessary (or recommended by the suppliers) to first let the blood settle under normal gravity, as suggested above (Seema Rai) but to be fair I heve never tried that or heard of it. It seems like a different method of using starch or dextran polysaccharide to sediment the red cells (under gravity in a tube at 45 deg from vertical) which will give you leukocyte-rich plasma containing granulocytes as well as mononuclear leukocytes, which I have used to obtain polymorphonuclear neutrophils.

Definitively I would not put the cells at 45 degrees as states Seema and not use 5000 x g.

What John tells you will work.

I agree with Jose Garcia-Sanz; You have to run your sample ~ 400 x g (~ 1500 rpm) @ > 37 C has recommended by sigma (histopaque-1077 - we were happy when working with this product); Has you already know at higher rpm your cells starts to lysis

I was isolating monocytes and T cells from human blood regularly.

I found that for the T cells: Lymphoprep from Axis Shield was the best reagent: http://www.axis-shield-density-gradient-media.com/Leaflet%20Lymphoprep.pdf

For monocytes, or isolation of PBMC from small amounts of blood, Ficoll-Paque seemed to work better: http://www.youtube.com/watch?v=fwnsHcL-wxg

We centrifuged at 1,500 rpm (around 300-400 rcf I think) for 30 minutes at room temperature and got a good separation with little platelet contamination .

From more than 10 years I do isolate on Histopaq 1077 (Sigma-Aldrich) or Ficol-Paque leukemic mononuclear cells (CLL). You have to add 2 volume of reagent and very slowly on Histopaq add 3 volumes of blood. Try do not mix blood with Histopaq and centrifuge your probe for at least 15 minutes/ 1500-1600rpm. In plasma you will see a usually white mono layer with mononuclear cell fraction

It is really simply.

You can separate it just by centrifugation at different rates because all of them have different densities

Hi,

I would like to add following comments:

- Ensure that you do process the bloods within eight hours of collection for optimal results using biohazard cabinet and sterile environment

- Aliqout plasma after initial spin, and the balance of the bloods mix with RPMI, then carefully aliquot on top of the Ficoll you have already put in your tube ( make sure no bloods will reach the bottom of the Ficoll as RBCs will be considered a debris and are useless for what you would want to achieve-we achieve this following this method- tube is straight and our pipette touches the side wall of the tube when releasing bloods to flow in).

- spin the Ficoll and bloods for 30 minutes on 1500RPMI and remove your "cloudy layer"(you can remove supernatant on the top and discard pipette, then you use sterile pipette to remove this layer)

- purify your "cloudy"aliquot with 10 ml RPMI (culture media)

- If you would like to obtain cell count I would recomend you have a dilution of 1 in 4 of the final pellet with RPMI. In my experience you should have high count of leukocytes where other WBC count would be negligible.

- As for final aliqoting and longer storage, following is important: prepare 800 microliter of foetal (or bovine) calf serum mixed with 200 microliters of DMSO. Your final aliqout should be placed into MR Frosty to ensure gradual freeznig (1 degree Celzius per minute). When you freeze it to - 80 transfer your aliquot to liquid nitorgen storage(-190).

DMSO is usually added when you would like to be able to freeze/thaw multiple times without loosing much of the cell viability.

Ficoll media we are using is GE Helathcare- BioScience.

I hope this answers your question.

Blood was collected aseptically from individual in heparinised vials.

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Blood was diluted 1: 1 with HBSS (Hanks’s Balanced salt solution)

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Diluted blood was layered on Ficoll in 1: 2 ratio.

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Centrifugation at room temperature at 550 RCF for 20 minutes for separation of buffy layer.

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Mononuclear lymphocytes and monocytes were recovered at the ficoll plasma interface where they from a white buffy layer.

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Cells at the interface were aspirated without taking out too much of the Ficoll as it leads to granulocyte contamination.

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Cells were washed in HBSS at 450 RCF for 10 min.

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Supernatant was discarded and pellet was taken.

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Cells were washed in HBSS at 400 RCF for 10 min.

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Supernatant was discarded and the pellet was dissolved in 5% CRPMI. (RPMI with 5% FCS and 1% Antibiotic antimycotic and glutamine).

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Cells were diluted 1: 10 in Trypan blue and 10 µl of this dilution is loaded in Neubar’s chamber and counted under microscope.

Cell count/per ml = Average no. of cells x volume of cell dilution x 104 x 10.

Just to add a few very basic but well known comments to above inputs:

1. Lymphoprep/ (Ficoll Hypaque)   ought to be at room temperature and not cold when you layer diluted blood onto it.

2. After centrifugation , quickly remove the cells at interphase and add any dilution medium for washing as density gradient medium is supposed to be cytotoxic.