Hi,

I have been doing competitive HSC transplantation to see peripheral blood chimerism up to 20 weeks post transplantation. I am double sorting HSCs (Lin-, Sca1+, Kit+, CD150+, CD48-) from CD45.2+ donor mouse by flow cytometry and injecting 50 HSCs with 300,000 helper marrow CD45.1+ cells into irradiated CD45.1+ recipients.

Although I tried several times to optimize it, I couldn't get rid of variability of my peripheral blood chimerism data. Among 10 recipients of the same injection group, I have 2-3 mice showing 40-60% CD45.2+ engraftment and the rest is ~5%. I confirmed the radiation regime is lethal at 14 days. I am doing retro-orbital injection into recipients under isofluorane.

Where might be the source of variation? What should I check further?

I have been using the same syringe for injecting into 10 recipients back to back, should I use individual syringes for each recipient from the same cell mix?

Thanks in advance,

Ozge