I'm trying to inject b lymphocytes in 2-3dpf embryos, I've tryed both in the yolk and in the duct of Cuvier, but cells always clump in injection needles, making it a nightmare.
I've started from 2,5x105cells/ml, but the situation did not improve even at lower concentrations or by adding EDTA or 1%PVP.
another question: is there any non-fluorescent dye that can be used to track liquids injection in the embryos?
any suggestion will be precious,
thank you in adavnce.
Hello,
You didn't say how you pull your needles or what pump apparatus you are using.
To transplant cells you need an oil-filled needle that is drawn to a large opening (similar to a patch-clamp needle) and a slow, positive displacement pump (such as a Hamilton syringe). See the Zebrafish Book for cell transplant methods:
https://zfin.org/zf_info/zfbook/chapt5/5.7.html
Hi, I'm injecting only into the pericardium of 3 dpf fish and I resuspend the cells in PBS containing DNase I and heparin at a concentration much higher than yours (5 x 108 cells/ml) but I have similar clumping problems. I didn't try to decrease the concentration at the levels you use, so it might be hard to do a comparison.
I think the best option is to follow Elizabeth E LeClair's suggestion.
Regarding your second question, I used to inject MOs, RNAs or transgenic constructs with phenol red as non-fluorescent dye to track liquids injection.
Hi Michela, try to use the Hanks buffer to diluite the cells before injection, this should avoid the formation of clamp in while, anyway is critical also the system that is used for injection, best air than oil in my experience.
About the use a non-fluorescence dye I agree with Matteo for red Phenol, but this unfortunatenly don't mark the cells but only the suspension.
Good luck!
- Badges
- Science topic
- Similar topics
- Phytochemistry
- Extracts