I'm not sure if the markers mentioned cover all possible myeloid cell types? Moreover, what about non-myeloid and non-melanoma cells that are not being excluded when you only use these 2 markers?

Not exactly sure what your question is, but Alexa Fluor 488 will cause spectral overlap in the PE channel. So you would have to include single stained controls to be able to calculate the compensation needed. Moreover you could consider the use of isotype controls.

Hi, as proposed by Gerco, the best negative control would be to include a tube where you replace your target-specific antibodies with isotype control antibodies labelled with the same fluorochromes. In practice, running such a control for CD11b is not absolutely necessary as you should not have any problem to gate the positive population (CD11b is usually bright). Nevertheless, as you decided to use CD11b coupled to Alexa 488, you should be careful about the autofluorescence of your cells in the Alexa 488 chanel (which is particularly significant on myeloid cells). In any case, an unstain control will help you to place your gate.

For the second staining, tyrosinase, it will probably be a little bit more tricky as you will do an intracellular staining I guess ? For intracellular staining, it is really useful to use an isotype control antibody to really evaluate whether your specific staining is real or just background due to cell permeabilization.

Good luck

Cheers

Metastatic myeloid cells in the lungs?? It sounds complicated because of the kind of tissue (lungs have a high density of blood vesels which generates the posibility of contamination of blasts coming from the blood, in such a case different kind of cells could be CD11b including NK cells). It would be better if you could identified solid myeloid tumors (myeloid sarcoma) and not only cells in a fluid. In such a case you should perform the diagnosis of myeloid sarcoma as recommended by the WHO classification of lymphoud and myeloid neoplasms 2008. If you are planning to identify cells in a fluid, I think it should include at least CD34, CD45, CD117, MPO, CD33. CD13, CD15 markers in order to better identified populations of myeloid origin (mature or immature). But again, it depends on how specific you need it. It is difficult to talk about "myeloid metastases" in the lungs when many cells CD11b+ are coming normaly from the blood. But there are many publications about flow cytometry and the minumun panel required for myeoid lineage.

Gerco,

CD11b has been used as marker for bone marrow derived cells committed to the myleoid lineage. The majority of myleoid cells keep the expression of this marker after differentiation into the many possible populations. This marker is suitable for the cells I am interested on.

Regarding the overlap, Alexa fluor 488 has an emission wavelength of 519 whereas PE has 575, so they are more than 50nm apart, they can be used together. However I am aware that I need to be very careful when analyzing my data as I might have some overlap, so I am including single stain controls.

I liked what you said about the negative control. I was thinking to use a carcinoma cell lineage which I know will not express neither CD11b nor Tyrosinase.

Bertrand,

The staff from the facility I am going to run my experiments said I do not need the isotype controls, but I disagree and just for safe I will run this control. Anyways, thanks for let me know that isotype control for CD11b it is not crucial. The CD11b I am going to use is an IgG raised in rat. Do you think I could use an Goat anti-Rat IgG Secondary Antibody Alexa Fluor 488 conjugate (which I am currently using for immunofluorescence assays) as isotype control? Or the Isotype control necessary has to be a given conjugated antibody against an antigen that is not normally expressed in the target tissue?

You are right, the tyrosinase staining will be a little bit tricky, because it is intracellular and require fixation/permabilization. I know these procedures might affect the some epitops and consequently the binding of the antibody. But I have already check with the company and the CD11b it is not affected by permeabiliziation (the Tyrosinase of course, is not affected since the protocol requires permeabilziation).

I got confused about the isotype controls for the Tyrosinase antibody. You said that would be useful to have isotype control for intracellular staining. However some companies websites suggest to not use isotype controls for intracellular staining, for example: http://info.ebioscience.com/bid/68300/3-Choices-to-Make-Before-Intracellular-Staining

Erick,

Thank you for you comments, but I think you did not understand my goal. I am not going to detect metastatic myeloid cells in the lungs, only myeloid cells and metastatic melanoma cells in the lungs. I know that just asking questions here without explaining the context of my project might be a little confusing.

HI Juliano

Usually the best isotype control would be an antibody of the same specy, same isotype and same fluorochrome as compared to your target-specific antibody. If you don't have anything else than a goat anti-rat coupled to Alexa 488 you can just try it ; but in your case the best would be a non-specific rat IgG coupled to Alx488.

Concerning the intracellular staining, I agree with what ebioscience says ; nevertheless for some intracellular antigens you can not switch on or off their expression just with a treatment... so it is difficult to use a non-treated sample as a control. In addition, I though that the isotype controls recommended on their data sheets were choosen based on the fact the antibody / fluorochrome ratio were similar between isotype and target specific antibodies... but apparently it is not the case. Anyways, for your tyrosinase I would try an isotype control ; you could also include a cell line that does not express tyrosinase...

When I do intracellular stainings, I usually stain membrane proteins first, they fix/perm, they stain intracellular antigens. But of course if the binding of your antibody is not affected by fixation, you can stain everything after fixation. Actually everything depends on which type of fixative you use.

Last thing ; Erick mentionned an important point. Before removing your lungs with metastases you should flush out blood if you really want to analyse myeloid cells that infiltrated lungs and not include myeloid cells that are present in the blood. Lung is a highly vascularized organ so if you are not flushing blood your samples will be contaminated with blood myeloid cells. To flush out lung blood, make an incision in the left atrium and then inject a few ml of PBS in the right ventricle.