My protocol to get PBMCs from mouse blood is a bit crude but simple.

Transfer the blood to a vial with 9 ml distilled H2O and mix by inverting a couple of times (this will kill the red blood cells).

Immediately after mixing add 1 ml of 10X HBSS or 10X PBS. This will cancel the osmotic pressure of pure water and stop the lysis process.

Spin down, discard supernatant et voilá.

Resuspend your PBMCs in whatever you need (staining mix, PBS, media, etc.).

(sometimes you get some blood clots, in this case, resuspend the cells in a volume of PBS or media and pass it through a mash)

My protocol to get PBMCs from mouse blood is a bit crude but simple.

Transfer the blood to a vial with 9 ml distilled H2O and mix by inverting a couple of times (this will kill the red blood cells).

Immediately after mixing add 1 ml of 10X HBSS or 10X PBS. This will cancel the osmotic pressure of pure water and stop the lysis process.

Spin down, discard supernatant et voilá.

Resuspend your PBMCs in whatever you need (staining mix, PBS, media, etc.).

(sometimes you get some blood clots, in this case, resuspend the cells in a volume of PBS or media and pass it through a mash)

Agreed.

Thank you for the answers!

Annemieke you are right: I do want to get rid of the granulocytes so I think lymphoprep is the easiest way for that.

And thanks for offering more help Dominic.

I will give the lymphoprep a try since we already have it available.

Another question just arised: I want to isolate mRNA for cDNA synthesis izn the end. Anyone knows if this is possible for individual mice or do I need to pool the blood of several mice to get enough mRNA?

I got the best results using histopaque with a lower gradient- around 1.07. It was a while back, so I don't remember the exact name of it. The histopaque for human PBMCs had a density of 1.84. This goes back to a larger size of human cells versus mouse.

You can also use Lympholyte-M (Cedarlane). One of the precautions that I always follow is to keep everything (blood, lymphocyte and centrifuge) at room temperature. Also turn off the brake on the centrifuge for a clean interface.

With regards to your second question, you can get upto 8ug total RNA from a million PBMCs as per this protocol: http://www.miltenyibiotec.com/~/media/Images/Products/Import/0002400/IM0002414.ashx.

But you will have to check and see how many cells you are able to get per prep.

I have been trying to isolate the PBMCs and sometimes is works, but I think 1.5 ml of blood is too little to get consistent amounts of PBMCs. (sometimes I do not get any, sometimes a few after lymphoprep centrifugation).

And I am wondering about the temperature component during the isolation. Vineeth said I should keep everything at room temperature. But a lot of people told me I should keep the blood on ice after sampling. Does anyone have more experience with either of it? Thanks a lot! 

Hi Navina,

I am doing CD4+ cells isolation from mouse blood. I am doing lymphoprep as described in the protocol; However, I did few changes in the buffer it is  PBS PH7.2+ 2mEDTA (this buffer recommended by Miltenyi Ficoll separation protocol) In addition to 2% FBS as advised by Lymphoprep protocol. I found it useful to add EDTA to prevent clotting and FBS to nourish the cells.  

First, I used 2ml of blood and got 10 millions cells after Lymphoprep separation. second time, I used 4ml and got 2X10e8 cells, which is good.  Yes not  all PBMC, but by FACS there was a nice uniform lymphocyte population with 99 viability.

My advice it: Not a lot of centrifuges. One with Lymphoprep. and Second for Wash exactly As advised by Lymphoprep protocol.

Here is the link for Lymphoprep and Miltenyi protocol

http://www.stemcell.com/en/Products/All-Products/Lymphoprep.aspx

https://www.miltenyibiotec.com/~/media/Files/Navigation/Research/Stem%20Cell/SP_MC_PB_density_gradient.ashxhttps://www.miltenyibiotec.com/~/media/Files/Navigation/Research/Stem%20Cell/SP_MC_PB_density_gradient.ashx

Hope that helps

Hi Navina,

At iQ Biosciences, we dilute the mouse blood, then perform a ficol spin.

If you want to save a lot of time, you could just buy too.

https://www.iqbiosciences.com/product/balbc-mouse-pbmcs-peripheral-blood-mononuclear-cells.

Please let me know if you have any additional questions.

Best Regards

Omar

https://www.iqbiosciences.com/product/balbc-mouse-pbmcs-peripheral-blood-mononuclear-cells/

Hi Pedro and Annemieke,

the method you use to isolate PBMC (with 9 ml distilled H2O) is able to isolate monocyes, too? Which is the starting volume of the whole blood?

Have you ever tried the optilprep?

Thank you. 

To isolate lymphocytes from mouse blood, as they are not called PBMCs from mice blood. The first thing to use is a Ficoll Paque PREMIUM with a density gradient of 1.084, which is suitable for the isolation of human PBMCs as well as mouse lymphocytes. Dilute the blood 4x in D-PBS. Layer it on Ficoll, then centrifuge. Before washing add 2.5mM EDTA and 1% FCS in the D-PBS, and centrifuge. One wash is enough. Second wash is not necessary as it may lead to low cell yield.

I am also trying to isolate PBMC from mouse blood. And I used the HIstopaque-1.083 to do density gradient centrifugation. The cells I harvested are limited（about 1 million every 4 ml blood) with platelet conntamination.  Is there anyone who can give me a detailed protocol or tell me how to improve the yield. 

I've been doing this for a couple months now. There are a couple things to note. You can minimize platelet activation but not stop it completely. Be sure to just mix the anti coagulated blood a few times; do not keep rocking it. Also, the size tube/volume you use to do the density gradient makes a HUGE difference in recovery in the band of PBMC. I use clear 2 ml tubes with a 1:1 of histopaque and blood then after that, pool the bands into a new, clean 1.5 or 2ml tube for all future washes (all in swinging bucket rotor). This works for me. But again, you'll never get rid of all the platelets. In order to do that, you'll need to then after the separation use the magnetic beads. Hope this helps.

Hi Betty，thank you for the suggestions. Would you like to tell me how many PBMC can be isolated from 1 mi mouse bloood? In fact, I try to test the cytotoxic activity of NK cells. And I found some people did  it with PBMC instead of pure NK cells. Is it reliable？

I just this week got samples. From my controls, I averaged about 4x10(6). I also am getting the blood through a cardiac puncture so I'm only getting about 700ul of total blood. 

Hi everyone, I need to isolate PBMC from mice next week, and it will be my first time trying it. I don´t have any kit for this protocol. I just have Percoll in my lab. Do you think that is going to work? Any suggestion?

I´ve been reading a lot of different protocols and I´m not sure how to proceed.

Thanks

The protocol we use involves a split of our blood sample since we also collect serum.  We split the initial blood draw into 2 tubes 1.) 1 mL Z serum MiniCollect tube (Ref#450470) and 2.) into a 1 mL Lithium heparin tube (VWR Cat#95057-295). You are correct about the mouse cell and the density. Thus we use Ficoll-Paque PREMIUM 1.088 (VWR Cat#95040-394) and follow the manufactures instructions for PBMC isolation. Please note: "Ficoll-Paque PREMIUM 1.084 can be used for isolating a broad range of human mononuclear cells including those of a higher density and for separating blood cells from mice or rats." Hope this is helpful, and good luck on your project.

We use 300 ul of whole blood in 1000 ul of Sigma ficol 1077 1 reagent and get excellent PBMCs which we use for PCR and genomic studies.

I think the easiest (and maybe the cheapest) way to isolate PBMCs from whole blood is by density gradient, e.g. histopaque 1077. I have used this multiple times with mouse and human blood and it works great (I've never used lymphoprep so I can't say much about it but I assume it works similarly). Other methods (magnetic/fluorescent separation) might get you a purer cell population but they are much more expensive and time consuming.

Dear all,

I am obtaining blood from a mouse (cardiac puncture) and then dilute 1:1 with PBS. I then use histipaque 1.077. I seem to get 4 million cells but only 50% viability. I am not sure what step I am doing wrong to have such a low %. When I isolate BM cells i get 90%+ viability.

For PBMCs we dilute 1:2 (1part to 1 part) with HBSS in a 1 mL Lithium heparin tube (VWR Cat#95057-295), which keeps the cells from clumping. It is important to collect the PBMCs as soon as the centrifugation is completed. We then wash our cells in HBSS to increase viability. Additionally you want to make sure your PBS/HBSS is Ca++/Mg++ free. Please note: For mice "Ficoll-Paque PREMIUM 1.084 can be used for isolating a broad range of human mononuclear cells including those of a higher density and for separating blood cells from mice or rats."

I need to Isolate PBMCs from a single mouse for effector T-cell preparation. I have Ficoll in the lab. and a protocol for human PBMC isolation with Dextran and Citrate Buffer to seperate the RBCs.

Do you think scaling the protocol down to 1ml blood volume will work?

I am highly tempted to do Pedro Veliça's suggested protocol.

Has anyone done something similar?