I’d like to share my experience with isolation and analysis of mouse hematopoietic stem cells by surface phenotype using flow cytometry.
Historically, a lot of methods was proposed for isolation and identification of hematopoietic stem cells (HSC). We can identify HSC based on surface or functional markers or using combined approaches.
The case I’m presenting below:
mouse strain: C57B6/J (WT)
age: 12 weeks
tissue: bone marrow
(click picture to enlarge)
backgate of LSK/CD34-/Flk2-/CD48-/CD150+ population:
(click picture to enlarge)
Abbreviations: LT-HSC – long-term hematopoietic stem cell; ST-HSC – short-term hematopoietic stem cell; MPP – multipotent progenitors
list of (anti-mouse) antibodies used:
CD34- Alexa Fluor 647 (eBioscience, cat #51-0341-82)
c-Kit- APC-eFluor 780 (APC- Alexa Fluor 750) (eBioscience, cat #47-1171-82)
Sca-1- PE-Cy5.5 (eBioscience, cat #35-5981-82)
CD150- PE-Cy7 (BioLegend, cat #115914)
CD-48- FITC (BioLegend, cat #103404)
Flk2/Flt3- PE (BD, cat #553842)
Lineage mixture primary (all from eBioscience): biotinylated Ter-119 (#13-5921-85), Gr-1 (#13-5931-85), Mac-1 (#13-0112-85), IL-7Ralpha (#13-1271-85), CD4 (#13-0041-85), CD8 (#13-0081-85), CD5 (#13-0051-85), B220 (#13-0452-85)
secondary: Streptavidin PE-TexasRed (Caltag/Invitrogen, cat #SA1017)
DAPI (Molecular Probes/Invitrogen, cat #D3571)
Instrument: BD LSR II
Software: FlowJo
*******************
Feel free to share your experience with us and discuss any technical details
PS: I was planning to share .fcs file, but it’s too big to handle online (=171Mb)
Related posts:
{ 1 comment… read it below or add one }
Dear Dr. Bersenev,
My name is Filipp Savvulidi. I am student in FACS and Image Analysis Laboratory, Charles University at Prague, Czech Republic. First of all, thanks for share your experience. In our laboratory we are doing same analysis of murine BM HSC by using flow cytometry (FACSAria II under DIVA soft). Our routine gating strategy a bit differs from yours (we gated out first “Lin neg” cells (it is about 5 percents cells of singlets accordingly to our data), and then from “Lin neg” we hierarchically gate all other populations). Your strategy to gate “cKit pos / Lin neg” first gives better “resolution” and so – more accurate gating; from this it is very interesting to me to compare your strategy with ours. Please, may you share some .fcs files with us? Or may be you may share with us “exported experiment” from your LSR II (then I can see your gates after importing .fcs in our DIVA)? I will be very thankful to you for sharing. Of course, if you will have interest, I can share our .fcs with you.
Thanks once again.
Sincerely,
Fil Savvulidi.