50 ml tubes, fill in 15 ml Ficoll (18-23°C!), IMPORTANT AT WARM!!! than 20 ml of buffy coat blood.

Buffy coat dilution: 1:2 with PBS (without Ca/Mg, room temperature!) IMPORTANT!!! room temperature! IMPORTANT BREAK OFF!!!!!

We obtained 1x10^9 PBMC in the interface

.

Washing: Use PBS or Hank's buffer 50 ml tubes per Buffy coat, cnetrifugation with 350g 7 min. 3 times.COLD AT 4C IMPORTANT!!!!

what speed n temp. do u centrifuge ? Do you further isolate the monocytes ? and PBS you add is cold ?

Can you please share your complete protocol with me ? [email protected]

Dear Raghav,

Do it again:

50 ml tubes, fill in 15 ml Ficoll (18-23°C!), than 20 ml of buffy coat blood.

Buffy coat dilution: 1:2 with PBS (without Ca/Mg, room temperature!) + 3mMol heparin.

Centrifugation: 250g, 30 min., room temperature!

Resuting in 5x10e8 - 1x10e9 PBMC in the interphace.

Washing: Use cold PBS + 2mMol EDTA, three 50 ml tubes per Buffy coat, cnetrifugation with 350g 7 min. 3 times.

Good luck!

Thanks for your reply Helmut, will do it as instructed by you. Thanks once again.

Quite okay with Helmut Jonuleit. But I am curious about Heparin and Ca2+. If you already have heparin in blood bag then why do you need to add heparin again?

What is the effect of Ca2+/Mg2+ in PBMCs isolation?

@ Oberoi: during centrifugation try with break off. Also try 400Xg for 30-40 min.

Dear Muhammad,

Sorry, little mistake. I mean PBS without Ca2+/Mg2+ to prevent clustering. Therefore, we add also additional heparin with good success.

50 ml tubes, fill in 15 ml Ficoll (18-23°C!), IMPORTANT AT WARM!!! than 20 ml of buffy coat blood.

Buffy coat dilution: 1:2 with PBS (without Ca/Mg, room temperature!) IMPORTANT!!! room temperature! IMPORTANT BREAK OFF!!!!!

We obtained 1x10^9 PBMC in the interface

.

Washing: Use PBS or Hank's buffer 50 ml tubes per Buffy coat, cnetrifugation with 350g 7 min. 3 times.COLD AT 4C IMPORTANT!!!!

Aggregation is the main issue here. Aggregation leads to increase in mass and hence it doesnt form layer within ficoll but sink deeper and settle as pellet. Diluting the buffy coat is the first step to reduce aggregation. Ca2+/Mg2+ free PBS and heparin further help to reduce aggregation. Importantly what density of ficol are u using? See the figure for the various layers you get before and after centrifugation in the following link (Histopaque gradients are the best for this purpose)

http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/General_Information/1/1119.Par.0001.File.tmp/1119.pdf

This twin gradient is required to separate granulocytes from PBMCs (if you want to collect both PBMCs and granulocytes as separate isolates, otherwise just use Histopaque 1077 alone so as to get only PBMCs....if you use Histopaque 1077 alone, the granulocytes will sediment with RBCs as pellet). Finally, do not overload the tubes with blood. Overloading will cause merging the layers after centrifugation. Of note, you should get a white thick paste like layer after centrifugation for PBMCs. If you dont get white layer, that indicates your buffy coat dilution with PBS is not enough. Good luck.

I have to concur that the biggest issue with your protocol is probably overloading. Easily solved by dilution with PBS or Hank's (preferably with some EDTA or another anti-coagulant). For leukapheresis packs I have frequently run 40 mL products diluted 1:1 with PBS/1mM EDTA.

For every 50 mL conical, put 15 mL Ficoll (well-mixed and at RT) then GENTLY layer the diluted buffy/cells on top. If you layer poorly and mix the interfaces, you will not get separation or poorly defined cell layers. Also be very conscious of centrifuging at RT, without brake and having a well-balanced centrifuge. Both of these affect the quality of the cell layer forming atop the FIcoll.

In addittion, get the sample as fresh as you can. 48h old buffy's tend to form aggregates easily.

good luck

We use this conical blood sample and get a lot of cells. We always diluted 1:5 in Versene before running it through Ficoll, which is basically PBS with added EDTA.

Good luck

We process a LOT of Buffy coats. We do everything at room temp. Dilute 1:1 with PBS. Gently layer 20 mls of this diluted Buffy on top of 20 mls of Ficoll in a 50 ml conical. Spin 30' at RT, 2000 rpm with the BRAKES OFF. If brakes are on you will not get a PBMC layer. The Ficoll we use is VWR cat# 80502-472.

Thank you all of u for ur valuable comments :) I really appreciate that. Thanks once again

Three tips:

Do all steps at room temperature!!.

Use Hanks or PBS w/o Ca +2and Mg+2. Cell aggregation is the main culprit for a non proper formation of PBMC layer.

Brake off.

Good luck

our protocol is as follows (all at RT)

1. BC is shrinked to about 75ml from a 500ml donation, add about 85ml PBS w/out

2. 10ml Ficoll in each of 4 50ml Falcons (I pipette ficoll directly out of the fridge bottle, by the time you are finished layering the blood it is as RT anyways), layer 40ml of blood in each

3. Spin 2800rpm for 15' break off

4. (lyse) wash with PBS w/out 1700rpm 5'

5. (lyse) two washes at 800rpm 5' (slow spin to get rid of the platelets).

Resuspend pellet in 15-20ml. I usually get between 250-500 million PBMC per buffy.

Good Luck!

Lots of good suggestions above. But, most of them are for blood. For an leukapheresis product dilute the product 1:4 with HBSS. Also depending on the age and source of the product the PMNs can degranulate and become hypodense. If this occurs you will have the Segs with the mononucs. I have no good solution for this, just frustrations. Additional potential problems experienced includes platelet aggregation (heparin is solution) and depending on product age, DNA from apoptotic/necrotic Segs. Add DNAse prior to processing. If you have this problem you will know about it by gross observation. I call it a hairy golf ball that appears in the product.

Temp of all products must be room temp, this includes the F/H, HBSS and product. Brake off. Layering as discussed above is critical and can be tricky the first few times. Ignore comments on rpm and use the correct G-force as rpm are centrifuge dependent. It is assumed that you are using a swinging bucket rotor .

Good luck!

The biggest issue with your method is the way you use Ficoll. The dilution with PBS should be 1:2 times, 10 ml of PBS with 20 ml of blood. Then in 50 ml tubes, add 15 ml Ficoll and very slowly add 20 ml of blood previously diluted. Then the centrifugation is a very important step. It has to be a ROOM TEMP and no brake at all during 30 min. You will get the buffy coat without any doubt.

I have purified high numbers of PBMC following similar or the same protocols mentioned. In some cases, I stimulated T cells from BC and found a much lower response (per million cells) than the same conditions for T cells from fresh blood. I.e. buffy coat production techniques alter the white cells, although I do not know how.

The step number1 should dwork why you said i tfailed?

May be because I did at 4 degrees.

I had also problems with Buffy Coats. They were not due to the protocol (similar than your) but the the Buffy Coat quality. Usually they give you not fresh samples, since legally they have to check for the main viruses. Find out how fresh if the Buffy you get. Probably many cells have dead....

Absolutely true. About 1/10 BC I get contains PBMC which are very sticky and hard to isolate, sometimes with in-transparent serum above the layer. If you always have problems asked your hospital for a fresh BC or draw blood from one of your colleagues to cross check whether its the blood quality or your protocol.

Dear Jordi, dear Sander,

Quality of buffy coat preparation is always a problem. However, it is not necessary to use old buffys after inspection of potential infections. We use the buffys directly after preparation as "potentially infected material", virus checks in the blood bank are "in process controls", results come later. This is in agreement with the "Good medical practice".

Some tips:

a) Use hand-hot buffy coats, talk with your blood bank. In winter time use boxes for transport to your lab to prevent cooling.

b) For PBMC isolation: You can store isolated PBMC in RPMI+1-2% plasma overnight at 4°C without any problems but not the buffy coat itself. However, storage of buffys at room temperature over night do not influence PBMC isolation significantly in our hands.

In conclusion: Not the age of an individual buffy coats might be very important, preparation and storage is more essential.

50% dilution of buffy coat with PBS is important before ficoll step.

I always use Histopaque 1083 and I have not diluted the blood; but I work with 3mL of Histopaque and 4,5-5mL of heparinized blood. You should centrifuged the samples at RT and, if possible, without accelaration and break for avoiding mix of bands.

I use the same protocol of answer 1 and of Gabriela Gorelik and I obtaine 5x108-1x109 PBMC

no my question was not related to the temperature which is pretty irrelevant . You said it did not work. what is it wrong with what you get on the Ficoll ?

If you are more specific i could help solve your problem

We usually use whole blood for the isolation of granulocytes but I have just written down the protocol we would use from the buffy coat stage to the isolation of PBMC,. We would usually use percoll too but I would assume that the ficoll is the equivalent. I hope it will maybe be of some use.

1. If 20/30 ml of blood was taken - top up to 40 ml with 1 x PBS and then top up to 50 ml with 6% (w/v) dextran in 1x PBS. Invert gently 10 or so times to mix (Causes RBC to stick together and sediment quicker). (If more or less blood, just make changes accordingly).

2. Leave for 25-30 mins – cap unscrewed to let air in.

3. Take the lighter red upper layer and transfer to another tube-centrifuge for 5 min @ 300 G, WITH BRAKE!! (Leave about 1 ml above the boundary before taking the rest with a pipette, top off with 1x PBS to 50 ml before spin).

4. Should get a red pellet with a little white above. Remove supernatant until where it goes conical at the bottom of the falcon tube ~ 3ml left. Re-suspend pellet by rocking gently and pipetting up and down a few times.

5. Make 68% percoll (6.8 ml percoll in 10 ml (1 ml is 10x PBS, rest is water). Slowly pipette a drop of the pellet on to the percoll at a steep angle and then release gently. When a layer starts forming, increase the angle and pipette a little faster until there is a nice clean interface at 10 ml. Centrifuge @ 700 G for 20 mins NO BRAKE!!

6. Collect the top PBMC layer to a new tube and top up with 1x PBS

Thanks Stefan :)

Franco, the layer of PBMC was not good enough. I have to pipette the layer several times in order to get it completly out. And later when I spin it down, the mononuclear cell number was 10-50 million, which is actually less from a Buffy coat, as futher I have to isolate the monocytes from then which is further too less.

Detailed protocols with many tips are also available at:

http://www.miltenyibiotec.com/download/protocols_sample_preparation_en/1396/SP_MC_PB_density_gradient.pdf

and:

http://www.helmholtz-muenchen.de/fileadmin/Immunmonitoring/pdf/PBMC_Isolation_Cryo_Thaw.pdf

Always try and use 'Buffy Coat' from Freshl Blood. The important thing to remember is always 'dilute' the buffy coat to a cell count which matches the blood count of a normal person. This can be done using RPMI or PBS. Then load on Histopaque 1077.

Make sure that the buffy coat is from fresh blood (and not from blood drawn the previous day), you can dilute 1:5 no problem, and do it at room temperature take into account the mix well coments, do not use ca++ nor mg++

Thanks all of you :) Things are going better now. Thanks :)

Dilute buffy coat 1/2 in PBS which will give you a volume of 140ml. Bring to 150ml so will have even amount to layer over ficoll.Layer 25ml over 12.5ml ficoll in 50ml tube. Do your usual spin at 300-400g at room temperature for 25 min. Remove and pool PBMC layer. At this point you will have tons of platelets so will have to do several low speed spins until supernate is clear. Contact me if you need more detailed protocol.

Consider the cell source. If it is coming from a cancer patient, you can have a high frequency of immature myeloid cells. Similarly, if coming from a mobilized patient you can also have a high frequency of immature myeloid cells. These cells are hypo-dense and result in myeloid cell contamination of the mononuclear cell layer. Regarding aggregation: this can be due to platelets or DNA from dead cells. At times, we find the addition of DNAse to be useful (critical). For overnight storage of apheresis products, we dilute 1:2 or 1:4 and store at 4C. Blood products for stem cell infusion or vaccine manufacturing are normally shipped at 4C.

I firmly agree about using DNAse. I am just adding it in all my protocols (especially if I am working with thawed cells). It can really make the difference, mostly in functional tests.

Buffy coat older than few hours or blood drawn under vacuum like vacutainer system (syringe with a 21G needle is better) often show DNA related clamps after ficoll gradient.

Dear Raghav,

Please follow this technique. Collect 50 ml of blood and place it in green top heparinized tubes. The blood should be slowly drawn using the butterfly needle ( 0.8 X 19 mm X 305 mm) without vacutainer into syringes. The blood is then placed in 5 heparinized tubes (sodium heparin 143 USP Units). Dilute 1:1 with PBS, and place 20 ml of blood onto 10 ml of Ficol (Cellgro). Centrifuge at 1600 rpm for 30 minutes. Avoid brakes on the centrifuge. Peripheral Blood Mononuclear Cells (PBMC) were recovered, pooled, washed, and resuspened in growth medium. The recovered PBMCs may be counted using coulter counter and used accordingly. I hope this helps.

you may try to use this protocol (page 2 in the pdf). It woks with an alternative strategy, simply by flotaton through a low density barrier.

http://www.axis-shield-density-gradient-media.com/Isolation%20of%20blood%20cells.pdf

Use a fresh buffy coat. Dilute with RPMI 1:1. spin with lymphoprep 2:1 at RT 800 g 30 mts would work. lalitha kabilan

Never ever use cold Ficoll! It changes the viscosity and it won't work. Also, when centrifuging, turn off the breaks of the centrifuge so that it stops slowly. As others here stated, 1:5 dilution seems a little high. 1:2 with PBS (without Mg, Cl) or HBSS should be good enough. You can even use regular medium, it doesn't really matter, even if the protocols say you should use PBS. The crucial factor is that the Ficoll is at room temp. I did over a 100 of such separations in various labs and it always worked.

These are all good suggestion. If you can you can, also try NH4Cl lysis. As this process would yield all the WBCs. Please find the details in J Autoimmunity, Dias et al 2013

Is it a centrifuge blood without plasma. In this case Buffy coat cells will have lots of aggregates and need to really dilute it with 1x PBC and shake gently to release cells. Allow this mixture to stand for 2-5 minutes and supernatant cells load it on Ficoll gradient and spin for 30 minutes at 3000 rpm in cold centrifuge. I thing you will get less yield but it will be pure PBMCs

DR. PRAVIN D. POTDAR

May I ask what would be the pH of PBS used for blood cell isolation?

Have you considered purchasing cryopreserved PBMCs? Will save you time and allow you to avoid these technical issues you are encountering. At iQ Biosciences, we routinely isolated PBMCs from buffy coats. I would be happy to send you our commercial protocol ([email protected])

Hi Omar Whould you be able to send your commercial protocol for me as well. It would be a great thing as we always bit up and down with in house protocol for PBMCs isolation.

Thanks in advance, my email is [email protected]

I dilute blood e PBS 1:1 and I think it works. Why do you dilute it 1:2? I would like to know. Also I overlay blood twice the amount of Ficol? For ex, 15 ml ficol overlaid e 30 ml diluted blood? Is that overloading? I would appreciate your reply.

Try dilution in PBS 1:1 (1/2), 25 ml of diluted blood on 15 ml Ficol, 2000 rpm, 25 min, 20°C WITHOUT break !

It is how I isolate PBMC since 3 years.

Good luck

Dried buffy-coat with few plasma could be a problem to separate viable PBMC from buffy-coat. If you can obtain buffy-coat without removing most of plasma it could be good for the quality of your  cells. Anyway we separate PBMC from buffy-coat by diluting 1:3 with PBS and centrifuge 30 min BREAK  OFF and  also low acceleration,  1800 rpm- Work at room temperature, (no 4°C) , ficoll density 1.077.

Good Luck

Paola

hi, Has anyone isolated PBMC from buffy coats post ON at 4°? Thanks!

This is common.  Optimally you would dilute it 1:1 with complete media prior to storing at 4C.  Sometimes it is critical, especially if the anti-coag is ACD to add DNAse to prevent DNA aggregation of cells from dead PMNs.

I isolated PBMCs from whole blood, at room temperature. At 4°C, it did not work. I don't know if it applies for buffy coat or not.  

I know its too late, but would like to comment on this. I have used blood bags devoid of plasma from hospitals. I have encountered enormous issues . the major culprit is the storage of those bags. it should not be refridgerated. the blood bag has to be maintained constantly at same temperature as that of room temperature. You have to instruct the hospital after removing plasma to store them at RT instead of cold storage. keeping the bag again in cold will lyse cells and the blood would be more sticky. if you need more advice on this i would be happy to share the protocol. that has worked good for me. always bear in mind that even after this the recovery of cells is not so good and you can also see activated PBMCs, probably due to serum starvation. hence if you could collect those bags immediately after plasma separation. you can avoid most of the problems.

Dinesh Kumar Parandhaman would you be able to share your protocol? We are also trying to use blood from blood bags for PBMC isolation. Furthermore, the blood bags can hold a maximum of 480 ml, however, if the bag contains less than this volume of blood the anticoagulant per ml of blood will be in excess (i.e. the blood:anticoagulant ratio will be altered) will this pose a problem to the PBMC isolation and cytokine studies thereafter?

Dear Jaclyn,

below is the procedure i followed. you would find some activated or distorted shaped cells which might die during subculturing.

Isolation of PBMC from Blood bank Buffy coats (blood bags)

We receive buffy coats after the blood has been processed for removal of plasma and platelets. However, there is still a small amount 5 – 10 ml of plasma that can be harvested from the blood bags. The blood bags should be kept in room temperature ( In India at 25 degrees) and should be centrifuged in the same temperature. The blood bag should never be stored in 4 degrees or in iced packs!!

After counting the cells are ready for culture and cryopreservation. For culture, resuspend cell at 10 million cells per ml so that calculations are easier Jaclyn Scheepers