I am new working with Flow citometry and I need to analize the proportions of eosinophils and neutrophils present in the mouse airways with 2 different treatments. My sample will be a wash with saline from the animal airways (commonly named broncheoalveolar lavage fluid or BALF). If someone has a protocol, I will be very grateful.
Hi Leo,
Do you fix your samples or do you use them fresh? Fixation will change the foward and side scatter and in my experience it should be easier without fixation because you can also use their size and granularity to identifiy cell populations. How many colours can you use? If your lungs are highly inflamed maybe you can use CD45 but for BALF I generally dont think its necessary. For the whole lung this is a very good staining protocol for myeloid populations: http://www.ncbi.nlm.nih.gov/pubmed/23672262
Both Neutros and Eos will have a high side scatter (granularity) but will be realatively small. The main population in the (uninflamed - I never did anything else) BALF will be alveolar macrophages - they are quite big (FSC) and also more granular (SSC) and highly autofluorecent.
Neutrophils are CD11b+, Ly6G+ , Siglec-F neg CD64-
Eosinophils are CD11b+ Ly6G- Siglec-F+ CD64-
AMs are CD11b-/low (but you will see a shift because of their autofl.) Ly6G-, Siglec-F+ and CD64+
Hope that helps! -Moni
Thank you so much Moni! I will try not to fix the samples. The machine is a FACSaria II (BD) with 4 colours. I expect eosinophilia or neutrophilia in the airways, depending on the protocol used. Do you have a simple protocol to share with the steps on how to manipulate/dye the samples? For example for Neu. I do not understand if I have to use (stain with) all the Abs (CD11b, Ly6G, Single-F, CD64) at the same time. I will be very grateful if you can help me. In my institution nobody works with FACs and even the machine is from a Core outside our institution, so it is hard to ask someone. Thanks for your time, I really appreciate it.
In principal: First you have to block the Fc-receptors - otherwhise you'll get a false positive signal (invarient chain of ABs is bound). We usually use TrueStainFcX from biolegend #101319. And then you use directly labelled ABs zB SiglecF-PE (BD 562068), CD64-APC (Biolegend # 139305), Ly6G-A488 (Biolegend 127625), CD11b-PerCP (Biolegend 101229) - of course you can choose other labels or shift them around! Of course you can also order the respective Isotype controls for your ABs.
First of all prepare a FACS buffer (1xPBS 2%FCS 2mM EDTA) and keep it @4°C (but it does not need to be sterile if you dont want to sort) Keep all your samples on ice, if you need a long time in the mousehouse you should ad some FCS in there, they will be happier :-)
For example; if I have 0.5x10^6 cells I would spin them down (ca 5min, 300-350xg, 4°C) and then resuspend them in 50µl FACS-Buffer containing the TrueStanFcX (in this case 1µl/50µl Buffer) - incubate on ice for 15 - 20 min.
meanwhile prepare your Master mix in FACS Buffer (for the amount of ABs look in the Datasheet), then you ad 10µl MM to each sample, vortex and incubate it for 25min on ice in the dark. Wash it 1-2x (ca 2ml FACS Buffer) resuspend in FACS Buffer & analyze.
Dont forget to do single stainings + neg control for your instrument settings!!
Do you need more information?
Poster from 47th Congress of Italian Society of Clinical Biochemistry (2015, October, Florence):
Detection of peripheral blood eosinophils by means of multiparameter flow cytometry
A. Marini1, G. Carulli2, T. Lari1, P. Sammuri2, C.
Domenichini2, S. Pacini2
1Lab.Osp. Versilia, Lido di Camaiore (Lu)
2U.O. di Ematologia, Università di Pisa
We report a simple method to identify peripheral blood
eosinophils by means of multiparameter flow cytometry
(MFC), using the association of CD157 plus FLAER.
These markers are used to detect clones of paroxysmal
nocturnal hemoglobinuria (PNH), which are
characterized by partial or total absence of
glycosylphosphatidylinositol (GPI)-anchored proteins.
FLAER is a fluorochrome-conjugated derivative of the
bacterial toxin aerolysin, which specifically binds the GPI
molecule and so is the best marker for recognizing PNH
clones. According to recent studies which provided
evidence about high sensitivity of MFC assays based on
FLAER plus CD157 combination, we added this reagent
in routine examination of patients with known or
suspected PNH. Flow cytometric assays were carried
out by a FacsCanto II cytometer. In the course of
analysis of cytograms, we noticed the presence of
variable percentages of granulocytes which were
positive for FLAER, but lacked CD157; since this
molecule is expressed on the surface of neutrophils, but
not by eosinophils, we hypothesized that the
simultaneous analysis of CD157 and FLAER might be
able to distinguish eosinophils from neutrophils.
Therefore, we studied a series of 31 subjects with either
normal or high number of circulating eosinophils, with (n:
4) or without (n: 27) PNH. The whole granulocyte
population was analyzed in a CD157/FLAER cytogram,
and granulocytes positive for FLAER but negative for
CD157 were expressed as percentage of CD45+ cells.
This percentage was compared with the percentage of
eosinophils enumerated by ADVIA 2120 (and confirmed
by manual counting), and a very high correlation was
found (r: 0.981¸ Spearman linear correlation test). After
setting a specific gate to include CD157-/FLAER+
events, followed by CD45/SSC back-gating, this
population was found to be characterized by higher
CD45 expression and higher SSC properties , as
expected in the case of eosinophils. To demonstrate that
the CD157-/FLAER+ granulocytic population consisted
of eosinophils, we performed sorting studies by means of
a Bio-Rad S3 Cell Sorter: the sorted CD157-/FLAER+
population consisted of well-recognizable eosinophils,
while the sorted CD157+/FLAER+ population was
represented only by neutrophils.
Sorry for the late answer! I have been out of the lab for the last 3 weeks! Thanks so much for your help Moni. I will ask if I have any doubt! Best Regards!
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