Normally, the whole blood is mixed with equal volume of PBS. Then the layer is made on Ficoll. We normally take the PBMC, but don't know whether that layer has your specific or target cells. We spin it at 1000-1500rpm. Optimum is 1000rpm. Guess this will help you.

Dear Mouna,

Hello & Good Morning,

You Can try a procedure that i do in my lab.

I usually extract Lymphocyte 3-5 times a week. I add equal volume of 1XPBS to the Actual volume of blood. The i add ficol at the base of the tube with a ratio of 4:3. (diluted blood is 4 ml then ficol will be 3 ml). Then I layer the diluted blood slowly on top of ficol.

i centrifuge at 1500 ROM for 40 Minutes with acceleration speed of 9 and Zero brakes (deceleration 0).

I get very good amount of lymphocytes. If i ever get RBC's alongwith the lymphocyte for any reason, then i add 1 ml of RBC lysis buffer for 1 to 2 mins and then i do the washing with PBS to get clear lymphocytes. I wish that it will work for you.

Thanks & Best Regards

The important thing is not to activate platelets (be sure the blood is well taken),

and avoid taking any of the platelet-enriched plasma layer.

I think is better you start from buffy coat, but you can adapt the protocol to whole blood.

Dilute 10-15 mL of buffy coat with PBS without Ca/Mg, 2mM EDTA buffer, to a total volume of 30 mL, at room temperature

Add the diluted buffy coat on top of your 15 mL ficoll solution

Centrifuge at slow speed (120 x g, 10 min, brake off, room temperature)

Remove 15-20 mL of the supernatant to eliminate platelets

Centrifuge at 350 x g (or your usual speed) for 20 mins, allow to decelerate witout brakes

recover PBMCs in PBS no Ca/Mg, centrifuge 400 x g 8 min, 2-8ºC

wash PBMCs in PBS no Ca/Mg, centrifuge 225 x g 8 min, 2-8ºC (see if you will need another wash).

Good luck!

Thanks to all of u for these interesting advices,

actually, i take 10ml of blood mixed with 10ml RPMI and i layer it slowly on Ficoll, then i centrifuge at 2500rpm (400g) for 25min, then i take the PBMCs, then centrifuge them for 20min at the same speed, (if i use less speed i get a very small amout of PBMCs) then i centrifuge them(2 times) between 1700 or 1500 rpm for 15 min,(but each time i have platlett in the bottom of the conical tube above the PBMC, and they get aggregated), i try not to use other solution to avoid any the activation of PBMCs during the separation, but i don't know if i decrease the centrifugation speed until 1000 would it be enough to have all the PBMCs? specailly, that i need from each 10ml of blood, at least 8 10^6 cells.

Mouna, first, check the density of your ficoll, for your purposes it should be 1.077 g/mL. If you follow the supplier's protocol you should not have yield problems, which is about 1-1.5 million/mL of human PBMCs (I guess you are separating human PBMCs). Platelet contamination occurs because of 2 reasons: by bad venopuncture procedure (agitation, anticoagulation, etc) and by mixing with the platelet-enriched plasma fraction at the time of PBMC layer extraction. Low temperatures (including solutions and centrifuge) also lead to plat activation and clumping. Be sure you use No brake in your centrifugations. Besides, I would not use RPMI to dilute your sample and instead, PBS or, if your problem persists, low cation PBS (no Ca2+ no Mg2+). The low speed centrifugation does not replace your separation step, it is simply a way to get rid of plasma before the whole processing. And is optional, you can do your process as always and take off all the plasma fraction, before isolating the PBMMC ring. Then you can wash thrice if it is necessary.

So, check the venopuncture quality; room temperature; no brakes; PBS washing or previous plasma separation. Hope to have been useful.

Perhaps you already have the attached protocol, but I'm uploading it just in case.

THank you very much, i appreciat your help, it's very useful

happy to help! by the way, my grandfather family is from Morocco!

Nice to know it :)

Hi,

After PBMNC separation for PLT removal, pellet shoul be resuspended in ca, mg free PBS in total volume of 10 ml and after centrifugation at 200*g for 10 min the PLT turbid the PBS and remove carefully with pasteur pipet.