In the next days Im gonna start with the preparation of decellularized extracellular matrix, for this experiment I found this protocol that in some parts is incompleted (marked in red). I don’t know if any of you have done this kind of experiment before, I will be really gratefull if you could help me out with it.
- After cells reach confluence, maintain the cells in serum free medium (supplemented with antibiotics S/P) for X days. How many days?
- The plates or dishes were placed on ice and washed twice in ice cold PBS.
- The cells were decellularized for X minutes with 0.25% Triton X, 0.25% sodium-deoxycholate (Merck) in PBS. How may minutes and which quantity?
Also set up controls for step 6 (actin and nuclei staining): brief treatment (30 sec; 1min).
- The isolated ECM was gently washed in PBS with 10 IU/mL DNAse (Sigma) followed by three washes in PBS. How many minutes?
- Morphological changes were observed under phase-contrast illumination using an inverted microscope.
- To verify the efficacy of this extraction technique, some extracted ECM samples were labeled for actin (phalloidin) and nuclei (Hoescht).
Thank you in advance