Xin,

Identifying 6 different immune cell types and additional subtypes within 1 tube is very ambitious.  The number of colors you can use depend upon the number and parameter of lasers and filters installed on your actual machine, though I do not work with an Aria, I believe it should be capable of at least 8 – 10 colors and if you can and do choose to try to stain that many you will have to be careful to work out the best combinations of fluorochromes that will work for your panel. Just because certain fluorochromes in theory should separate out nicely doesn’t necessarily mean they will, especially when working with such a large panel. Personally, I prefer to keep staining to 8 colors or less especially if I am looking for rare populations. Does your school/lab have a flow cytometer manager or someone who is in charge of the cytometer? If they do you can work with that person to figure out the exact setup for your instrument.

In addition to your desired markers you should also always include a live/dead stain to gate out dead cells, otherwise they can give you false positives, this will take up one of your colors.  For your specific panel I will start with the cell types I am most familiar with:

T cells – For T cells you really should use a T cell specific marker to ensure you are looking only at T cells I would use CD3 to gate for T cells and then use CD4 to look at the CD4+ helper T cell subsets. CXCR3 and CCR6 are not specific to T cells. Both are chemokine receptors and unsurprisingly are on a variety of immune cells.  In addition to T cells, CXCR3 is also on B cells, NK cells and Macrophages and CCR6 is also on B cells and DCs.  Even within T cells CXCR3 is also expressed on certain CD8 T cells. Therefore, you will need to first use T cell specific markers to gate those out.  After you have gated on CD3+CD4+ cells you can use CXCR3 and CCR6 to help identify Th1 and Th17, though you can also use intracellular markers too such as IFNy and IL-17 or T-bet and RORyt.

Dendritic cells can be identified as CD11c+ and MHCII+, but remember that these markers are also on other immune cell types too. Most people gate DCs by first gating for lineage- cells and then gating on CD11c … You can buy lineage antibody cocktails that stain for CD3, LyG6/Ly6C, CD11b, B220 on one color so you can easily discriminate them, but since you want to use these makers to also identify other cells as well a cocktail on one color may not be the best thing to use, unless you want to and can stain for multiple tubes.

Macrophages: (F4/80+, MHCII+) should work

Monocytes: (MHCII-, Ly6C+, Ly6G-) should work

Eosinophils: (MHCII-, Ly6C neg or intermediate, Ly6G-) should work

Neutrophils: (Ly6C intermediate, Ly6G+) should work

Using these markers would put your panel at 10 colors (Live/dead, CD3, CD4, CD11c, MHCII, F4/80, Ly6C, Ly6G, CXCR3 and CCR6 (or other Th1/Th17 differentiation markers, I prefer intracellular ones))

Also how are you isolating your cells? Keep in mind that extracting cells from intestinal tissues can be rough on the cells and may effect surface markers. We found that certain preparations of collagenase down regulate certain cell markers and we had to optimize our protocol with staining in mind. The Jove video below touches on that issue as well.

A few articles to consider:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3987771/ (monos, MO, PMNs, and eosins)

http://onlinelibrary.wiley.com/doi/10.1002/cyto.a.20859/pdf  (12 color flow panel strategy)

http://www.jove.com/video/4040/isolation-characterization-dendritic-cells-macrophages-from-mouse (jove video for isolating lamina propria and flow staining)

I hope that helps.

Hello Xin Wang,

If your experiments consider human tissue i could propose to use firstly a CD45 antibody, so as to exclude all not immune system cells from your analysis. Monocytes and macrophages are positive for CD14 and CD206 and also monocytes have high expression of CD33 whereas macrophages are just CD33 positive. A problem is that dendritic cells are also CD14 and CD206 positive, so maybe you will want to include CD123 as a negative marker for monocytes and macrophages. Of course i do not know the presence of dendritic cells in the colon and if it may cause a problem in your analysis.Now for the neutriphils, they are CD16 bright and CD33 positive whereas i think eosinophils are CD16 negative (not sure about that).

The gating strategy may be the following: First you gate only in the singlet events and consecuently distinguish immune system cells with a SSC vs CD45 dotplot. In that dotplot the several populations do tend to differ, e.g the neutrophils have intermediate CD45 and high SSC, the eosinophlis are just to the right of them and monocytes/macrophages have intermediate SSC and a slightly higher CD45 expression. Thereafter, you divide your populations in two subsets containing neu/eosin and monoc/macroph by drawing gates in the first dotplot and distinguish neutrophils as CD33 positive/CD16 bright, eosinophils as CD33 positive/CD16 negative, monocytes as CD14 positive/CD206 positive/CD33 bright whereas the macrophages are CD14positive/CD206 positive/CD33 positive. Here you can add a CD123 to exclude the dendritic cells.

Alternately, you take the CD45 positive cells and distinguish the population based only on the markers and not in the the scatter properties.

Of course you can exclude CD206 and CD123 if it suits you, and even include markers for the subtypes of macrophages so as to distinguish them better from the monocytes. An other solution is to add CD64 as an extra marker or a better subtitute of CD206, because the monocytic lineage is bright for it whereas the neutrophils just dim.

I hope this helps,

Best regards,

Nikos

There's a lot of research going on on this field ... I would do as follows (as alternative to Nikos - his solution would work too).

CD45 positive cells to catch the immune cells. From them you take the CD11b+ cells. This population you can decipher with the following antibodies against: Ly6C, Ly6G, Siglec-F, MHCII, F4/80.

Eosinophils scatter high in the side and should be SiglecF+. Neutrophils are rather easy to detect: Ly6Cmed, Ly6G+.

With the remaining cells: a blot of Ly6C against MHCII should give the "monocyte waterfall", which shows you stages of monocyte development to macrophages. Macrophages should be F4/80+, MHCII+, Ly6C-. Freshly arrived Monocytes should be CD11b+, Ly6Chi. Take generally care of DCs ...

Papers of Allan Mowat are great for that ...

e.g. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4169290/

Hi Nikos E and Florian,

    Thanks for you detailed information.

    Here is another problem, Considering the cell number in the distal colon, I need to quantify both Th1, Th 17cell, Macrophages, Monocytes, DC, Eosinophils, Neutrophils in one tube.

Here I want to use this gating strategy:

Macrophages:( F4/80+, MHCII+)

Monocytes( MHCII-, Ly6C+, Ly6G-)

Eosinophils( MHCII-, Ly6C-, Ly6G-)

Neutrophils(MHCII-, Ly6C_inter, Ly6G+)

DC( CD11c+, MHCII+)

Th1 (CXCR3+, CCR6-)

Th17(CXCR3-, CCR6+ )

  Does that fit for my purpose? It seems that I need to stain multiple antibody in one tube, and I use the  Aria II for flow cymentry, how many colors can I stain in one tube? Thanks!

Xin,

Identifying 6 different immune cell types and additional subtypes within 1 tube is very ambitious.  The number of colors you can use depend upon the number and parameter of lasers and filters installed on your actual machine, though I do not work with an Aria, I believe it should be capable of at least 8 – 10 colors and if you can and do choose to try to stain that many you will have to be careful to work out the best combinations of fluorochromes that will work for your panel. Just because certain fluorochromes in theory should separate out nicely doesn’t necessarily mean they will, especially when working with such a large panel. Personally, I prefer to keep staining to 8 colors or less especially if I am looking for rare populations. Does your school/lab have a flow cytometer manager or someone who is in charge of the cytometer? If they do you can work with that person to figure out the exact setup for your instrument.

In addition to your desired markers you should also always include a live/dead stain to gate out dead cells, otherwise they can give you false positives, this will take up one of your colors.  For your specific panel I will start with the cell types I am most familiar with:

T cells – For T cells you really should use a T cell specific marker to ensure you are looking only at T cells I would use CD3 to gate for T cells and then use CD4 to look at the CD4+ helper T cell subsets. CXCR3 and CCR6 are not specific to T cells. Both are chemokine receptors and unsurprisingly are on a variety of immune cells.  In addition to T cells, CXCR3 is also on B cells, NK cells and Macrophages and CCR6 is also on B cells and DCs.  Even within T cells CXCR3 is also expressed on certain CD8 T cells. Therefore, you will need to first use T cell specific markers to gate those out.  After you have gated on CD3+CD4+ cells you can use CXCR3 and CCR6 to help identify Th1 and Th17, though you can also use intracellular markers too such as IFNy and IL-17 or T-bet and RORyt.

Dendritic cells can be identified as CD11c+ and MHCII+, but remember that these markers are also on other immune cell types too. Most people gate DCs by first gating for lineage- cells and then gating on CD11c … You can buy lineage antibody cocktails that stain for CD3, LyG6/Ly6C, CD11b, B220 on one color so you can easily discriminate them, but since you want to use these makers to also identify other cells as well a cocktail on one color may not be the best thing to use, unless you want to and can stain for multiple tubes.

Macrophages: (F4/80+, MHCII+) should work

Monocytes: (MHCII-, Ly6C+, Ly6G-) should work

Eosinophils: (MHCII-, Ly6C neg or intermediate, Ly6G-) should work

Neutrophils: (Ly6C intermediate, Ly6G+) should work

Using these markers would put your panel at 10 colors (Live/dead, CD3, CD4, CD11c, MHCII, F4/80, Ly6C, Ly6G, CXCR3 and CCR6 (or other Th1/Th17 differentiation markers, I prefer intracellular ones))

Also how are you isolating your cells? Keep in mind that extracting cells from intestinal tissues can be rough on the cells and may effect surface markers. We found that certain preparations of collagenase down regulate certain cell markers and we had to optimize our protocol with staining in mind. The Jove video below touches on that issue as well.

A few articles to consider:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3987771/ (monos, MO, PMNs, and eosins)

http://onlinelibrary.wiley.com/doi/10.1002/cyto.a.20859/pdf  (12 color flow panel strategy)

http://www.jove.com/video/4040/isolation-characterization-dendritic-cells-macrophages-from-mouse (jove video for isolating lamina propria and flow staining)

I hope that helps.

Hello again,

I agree with Rose regarding the viability assessment dye. Maybe you should break the protocol in two tubes, one for the inate immunity cells and one for the T cells. In each tube you can use backbone markers to distinguish live cells and lineages such as CD3. In one tube the strategies to identify monocytes, macrophages, neutrophils and eosinophils mentioned above should do nicely. And in the tube for the T cells you could add extra markers for Th1 and Th17, possibly via intracellular staining like Rose elegantly suggested. I believe, regarding T cell subpopulations the more markers you have to consolidate what you are seeing the better.

Best regards

I agree with the above mentioned things. Do NOT put those stainings together - you will end up with confusion. Especially, if you are not experienced. Divide and conquer: Innate & T cells. For T cells I'd also do intracellular stuff like T-bet/RoRgT/Gata-2 + CD3 (very important not to catch ILCs) ...