Hi Guillermina!

There seems to be something there but in very small amounts, as I dont know the protocol it is very hard for me to say what could have gone wrong. I have also not been using histopaque, so I am not sure how similar it is with percoll, which is what I have been using. Are you sure you are layering the histopaque layers very carefully for the gradients not to mix? That could make a potential difference. I am not sure how you are preparing the histopaque, but if you are diluting it from its original density with HBSS for example, as I do with the percoll, you could dilute one of the densities with HBSS with phenol red, to see the interface of the two gradients more clearly and if they are mixing, then you may know if this is what makes a difference. I have posted a picture from a previous protocol I used to work with which used three layers of percoll and where one is with phenol red, and the top is the cell layer from the bone marrow.

I recently succeeded to isolate murine neutrophils with a protocol I find very consistent and easy, that only uses one density of percoll, it is the Mocsai labs protocol, which I have attached here. 

Good luck, feel free to write to me if you have further questions.

Teodora

Hi Guillermina,

I usually work with 3mL of Histopaque 1077 layed on 3mL of Histopaque 1119. Just remember to let histopaque on room temperature before use. After, I ressuspend my cells in 1mL of PBS an layed on top of Histopaque 1077. Just remember to centrifuge with low accel and break free. It is also important to centifuge at 20 -25 degrees celcius.

I use this protocol when purity is not crucial for my experiments, since this protocols give me average of 70-85% of purity. If you are going to perform some FACS experiments I think it is ok, since you can marker the cells with Ly6G and gated them.

If you are performing an experiment that purity is really important, I suggest magnetic separation that gives you a purity of 90-95%.

Good luck with your experiments,

Leandro

Dear Guillermina, I totally agree with Teodora Andonova. I also used percoll gradient and HBSS, etc. Another important point is the temperature (I tried to do everything, including centrifugation at 4C) and centrifugation speed (the speed of ascending and descending) and rotor (look here, table 3, how rotor may affect the quality of zonal and isopycnic sedimentation http://www.coleparmer.com/TechLibraryArticle/30 ). I would not say that the neutrophil separation/isolation is very easy technique, but if you will take into acoount all these tips, you should be able to get nice layer if neutrophils with the purity circa 90-95%. Good luck!

Thank you so much for your answers! I've worked with ficoll and peripheral blood neutrophils so I am kind of used to the layers and different densities. But, you're probably right, maybe I am mixing a little the layers, and I can't really tell because they are both transparent. I bought both Histopaque so I can't make any dilutions with Phenol Red Teodora :( too bad! your percoll looks beautiful!! 

Leandro I am doing exactly as you're saying, 20 degrees, no brake, 1ml of PBS on top of those 2 other layers...

I'll just have to keep trying... Thanks!!!

Hi Guillermina. I have had no trouble using the Mocsai protocol as in Teodoras attachment.

Also - and I am sorry, I know you have experience with layer centrifugation but I have done this myself once - are you sure you have the layers the correct way round? If not that would give you a single band at the PBS interface...

Hi Guillermina, I had exactly the same problem as you.  I tried everything in their troubleshooting guide and had a very clear boundary between the two solutions.  I could only get a clear halo of neutrophils when the cells were centrifuged for a long time or at high speeds.  This concerned me as i thought it might activate them so i ended up using magnetic beads.  More expensive definately, but my samples are really pure and thats what i need.   

Thanks Sherina! Just in case I also give up, what magnetic beads are you using? Thanks!

Hi Guillermina,

I am using MACS beads, from Miltenyi Biotec.  I got very frustrated with the Histopaque and spent a long time trying to get it right. I think its important to admit when things don't work out as it might save others time!

Best wishes

Sherina

Hi,

I did this protocol several times and I also got kind of like "frustrating halos". When got a result as you got I tried to figure out what was happening and then I propositally committed some mistakes like disturbing the interface or spinning down on 4ºC instead of RT to check it out.

- Mixture of layers: I cannot see any halo. (cannot distinguish the populations anymore) 

- Centrifuging at 4ºC: Same result as you. Clear monocytic/lymphocytic layer and spread neutrophils all over the gradient.

What I realized was that actually even when the lower halo is not as visible as the upper one, the neutrophils are dispersed from the lowest limit of the first halo downwards.

I checked the populations in the first halo and in the dispersed remaining layer and as I mentioned, the neutrophils are about 90% pure there. 

The first cloudy and evident halo are monocytes and lymphocytes which comprises the majority of WBCs in mice as opposite to human WBCs.

You can spin down the remaining content of the tube after adding the same volume of the buffer your using and you will see a great pellet there. They are majority (~90% neutrophils).

In summary, I do like this protocol when isolating from blood. But when working with BMCs I prefer much more the Percoll gradient, 5, 3 or one layer. In my experience they are much better than any method, plus, it is inexpensive compared to magnetic beads and there is the advantage of maintain them inactivated.

If you have an ultra-centrifuge available you can also use the continuous Percoll gradient protocol, which is also really good. 

Hope this could be helpful.

PS.: the protocol from Mocsai lab is exactly the same as commercial Cayman kit for neutrophils.

You guys are using ficol separation. In vivo we use 6% hespan. For myelocitic leukemias we do leukoreduction using a solution of trisodium citrate (hard to get a hold of, but hospital pharmacies have it), and add Hespan.  The RBC's and other precursors form rouleaux and we can harvest the granulocityc layer by centrifugation.  Attached is one of my illustrations that is in Henry, Textbook of Clinical Laboratory.

Formula for Hespan in Trisodium Citrate. 

This is from the FDA site. This is for human use. 

We mix this solution and prime the apheresis machine with it. We run this at a ratio of 13:1 (13 drops of whole blood for 1 drop of the mixture). Trisodium Citrate is a chelating agent. Ionized calcium will drop to almost nothing if not supplemented. In your situation I would not worry about it.  Please let me know if it worked. 

Thanks

Sergio