Hello,

During My Ph.D. I transplanted spermatogonial stem cells from GFP mice. I was able to visualize the GFP cells in the testes up to 4h post sacrifice. I was visualizing the cells in a discetion microscope equipped with a fluorescent light soucre (maximum magnification 80X) . You are saying that you do not have the equipment for immunostaining, do you mean that you do not have access to a fluorescent microscope ?

If not, have you tryied to use your fluorecent source to select GFP+ mouse with a disection scope ?

Thanks for your answer but there's been a misunderstanding : my lab is equipped for fluorescence microscopy and immunostaining, but this type of experiment would take too long if I need to look at many different types of organs from mice sacrificed at various time points. That's why I'm looking for more automated methods.

Sometimes depending on the construct you cannot detect GFP

+ cells directly by fluorescence microscopy and you have to enhance with an antı-GFP antibody staining, also quantification would be more difficult. I would recommend flow cytometry combining with stable markers for stem cells (if they are stable) and it would better if you use GFP stemcells and graft them into congenic mıce (eg. CD45.1 into CD45.2 mice).

Thanks. Should I perfuse the mice with saline before dissecting the brain, liver, lungs, spleen, bone marrow...? How should I dissociate them afterwards? Do you have a precise protocole for preparing cells from these organs? Should they be fixed or analysed fresh?

What about an ELISA against GFP?

No idea about ELİSAs, yes perfusion is required when you analyze organs. Dissociation requires enzymatic digestion and mechanical disruption, which enzyme to use depends on the organ; you have to check protocols. I know they are out there but only used a protocol for isolating leucocytes from testes. can analyze fixed, it does not affect GFP but make sure it does not affect other markers you might use.

Hi,

have you thought about using fluorometer (plate based)? This way you could use crude tissue dissociation and read the relative fluorescence in comparison to naive tissue.

Yes, I thought about it as well. It would be much simpler but I fear the fluorescence will be so low if I only have few transplanted cells that it won't be sensitive enough... I guess I need to test to know!

Seems a shame to use GFP cells in other methods of detection.

If you don’t use the GFP fluorescent properties,

I saw in some papers grafting cells of the opposite gender then tracking Y/X chromosome properties.

Flow cytometry should be easy enough to do with fluidic tissues, such as spleen and blood (I suppose bone marrow as well but have no experience in that).

Dissociating solid tissues into single cells suspension can be a pain, (I used the milteny's gentle MACS) you will need to adjust enzymatic protocol to each organ and there is a loss of cell viability in many tissues.

Best of luck

Ehud

Hi Isabelle, you did not mention which stem cell do you plan to transplant (HSCs?) Separated stem cells? The host will be conditioned prior to transplantation? How many cell will you transplant? After how long period will you sacrifice your mice? What GFP strain do you use ? (there are wast differences)

I have injected CFSE stained cells into mice, and taken out numerous organs a few days later to track where the cells went, this works fairly well. I'm not sure how long or strong your GFP signal would last after injected and left in the mouse for a while.

You would want to perfuse the mouse if you want to exclude the cells present in the blood that didn't actively track to that organ. Then mechanical dissociation (squishing with plunger in cell strainer) works well for spleen, bone marrow is easily vortexed once you plunge it out of the bone. Liver, lungs and brain will require enzymatic digestions (combinations of collagenase, hyaluronidase and or DNAse) as well as mechanical dissociation. Liver and lungs should go easily into single cell suspension after digestion with a syringe plunger in a cell strainer, but I'm not sure about brain.

All of this will take alot of work. If you plan to sacrifice even just 10 mice with 6 samples each, 60 samples without additional fluorescent tagging (with antibodies), this might take 3 people all day, a long day. And FACS acquisition the next day.

If your lab is set up for prepping organs for sectioning and staining, I would think that this would be easier and less time intensive, just because you can set your tissues in freezing medium or paraffin and make sections later, and stain for anti-GFP later.

I suppose it wouldn't be as quantitative though and would take a while to count GFP cells. Its just that with FACS, you could get to the end of your day and realize that the GFP signal was lost, have to go back and stain for anti-GFP and re-aquire everything.

If it were me, I'd do a trial run and just take spleen from a mouse a couple days after transfer and see if you can detect that by FACS.