2.7. In Vivo Wounding Assay

2.7. In Vivo Wounding Assay. 

To analyze the wound homing capacity of XtiSCs, the wounding assay was performed as described [24] with modifications. Briefly, stage 51 or older (around 3-week-old) X. tropicalis larvae were anesthetized with 0.01% tricaine (MS-222) and put into a Petri dish with 6% Ficoll, 0.1x MMR, and 0.1% BSA. 

In our later experiments we found out that it is not necessary to put tadpoles to 6% Ficoll but 0,05 x MMR + gentamicin could be used instead for the microinjection environment. 

Two hundred RFP-positive XtiSCs (40 nl) treated or untreated with CHIR99021 had been microinjected into larvae through dorsal blood vessels near the abdomen. 
 

We used pneumatic microinjector IM-300 from Narishige for the cell microinjection and oil microinjector IM-55-2 for holding the anesthetized tadpole. The IM-300 allows microinjection so as aspiration of cell suspension. It is good to use fresh aspirated cell suspension after 5 injected tadpoles. Here is a figure showing the holding the tadpole and position of microinjection capillary before microinjection into dorsal vein 
 

Just after microinjection, the distal third of the tail was wounded by #55 Forceps (Fine Science Tool). 

We just tweek the the tail in the last third with forceps.