Hi. If you are going to understand the HSPC populations in your knock out mice, you will need a guided next door or near by. There are multiple pitfalls to all of the analysis you are about to do.. Do you know someone nearby who works on hematopoietic stem cells?

are you using counting beads or how do you determine your cell numbers?

i have similar difficulties dealing with mice of different genotypes, maybe you could elaborate a bit more on some of these "pitfalls" you were talking about, Jonathan Keller? Thank you!

thanks for your answer. unfortunately we don´t have experienced people around (on our campus). we are at the very beginning of this project - of course our collaborators are experienced but still I wanted to start right now with the experiments and each kind of advice is helpful as you can imagine... The main part will be the transplantation experiments to check for hematopoietic reconstitution upon irradiation but I want to start more with functional assays: colony forming assays, cell cycle analysis, apoptotis, phosflow,... therefore I have to find my populations of interest first by FACS analysis

Hi Christian,

BM cellularity can vary greatly in ko, Tg etc mice, conditions influence is also common (if unsterile housing and mice are under some sort of inflammation you'll have feedback to BM to produce more cells hence cellularity will go up).

Also depending on mice strain you can see differences btw wt and ko (and also depending on the ko, what gene, what it does, etc): ko having more or less cell counts (absolute numbers) and/or %. I usually count cells on hemocytometer and then run flow: gate out DAPI positive then take Lin negative cells -> LSK (cKit+, Sca-1+) and then you can differentiate LSK with Flt3 vs CD34: "true" HSC (or long-term HSC) will be Flt3- CD34-, short term HSC will be Flt3- CD34+. And then use flow % to back-calculate absolute cell numbers.

My present supervisor published this paper few years, it may be useful to you to address different questions in mice BM: http://jem.rupress.org/content/208/9/1757.long

Hope this helps.

Best,

Miloje

Knockouts can affect gene expression. Thus, using markers as surrogates of biological readouts has limitations. Adding the fact that the KOs are smaller and presumably have reduced cellularity and potentially altered percentages of HSCs and progenitors, the quick strategy of flow cytometry may be misleading.

Although we perform the type of analysis that you are attempting right now. We perform flow cytometry analysis of HSC and progenitor populations to complement our functional assays - ie., bone marrow transplantation, LTCIC, CFU-C. If you are having problems with the flow, I would perform functional assays only including limiting dilution BM transplantations, serial transplantations and competitive repopulations. These are the gold standards because they give you functional readouts.

Good luck,

Bill

Dear Christian,

a quick way to get an idea if hematopoietic development is altered in your knock-outs would be to sort/enrich the c-kit positive fraction and then perform colony assays plating different numbers of starting cells. That would give you CFC forming units per 1000 c-kit+ cells. These are hard numbers and you can do your stats on them. I don't believe that there is any method that could reliably record a 0.3% difference in a biological value. And even if there were one, what significance would that have? If there isn't at least a two-fold change (+/-) I would think twice before following up that lead.

Just my 0.02$ - good luck

Robert

Tricky analyses to perform if you have no one to help you. I would read up on some papers and try it a few times with WT mice, then when you get the hang of it, do it with your KO mice. Concerning the questions:

1. Yes, infections (virus/baceteria) will affect stem cell numbers. It was shown that interferon will activate dormant stem cell, and there are many other papers along the same line

http://www.ncbi.nlm.nih.gov/pubmed/19212321

2. There are many ways to quantify stem cell numbers. On of the most established is % of xz population of total bone marrow cells. For LSK cells that would be in the range of ~0.1%. I appears that is what you are using, and that is what many other people are using.

3. Plating. I would suggest starting with a normal plating protocol starting with whole bone marrow cells. For some purposes sorting cells is advantagous, but if you are interesting in the % of colony forming cells within the BM, then plate normalized BM cell numbers into methyl cellulose.

Here is a protocl describing some basic techniques in a very basic way, but it might be helpful:

http://onlinelibrary.wiley.com/doi/10.1002/9780470942390.mo110088/abstract

If you are interested in a more in depth analysis of HSCs, this paper is what a lot of people use as a reference:

http://www.cell.com/cell-stem-cell/abstract/S1934-5909%2807%2900071-9

if you have any other questions, please do not hesitate to ask.

cheers,

Hi

10 000 nucleated bone marrow cells contains 1 repopulating units/HSC. http://www.ncbi.nlm.nih.gov/pubmed/12397098

Direct way for HSCs humbler estimation is to transplant you knockout mouse bone marrow in limiting dilution (3000-5000 of nucleated cells to single recipient) in compression with litter-mate control. You could estimate after the number of short term / long term repopulating mice. And calculate by ELDA software number of HSC http://bioinf.wehi.edu.au/software/elda/

The same quantification could be done for myeloid progenitors count in total nucleated bone marrow by limiting dilution. Mouse bone marrow cells contains 50-100 CFU-C per 10 000 nucleated cells. (it a bit varies with age and strain). To see difference with your control for myeloid CFU-C you could plate 1000 nucleated cells per dish in methyl Cellulose culture (MethoCult® GF M3434) http://www.stemcell.com/en/Products/Popular-Product-Lines/MethoCult/MethoCult-GF-M3434.aspx

Sorry, it is of course direct but a bit more expensive way in comparison with LSK or LSK150 estimation by FACS.