Hello Moleculardude, Hello kiki06
happy to hear news from you about my problem. At first many thanks for your suggestions, me too I think that the problem is during the perfusion, may be is not possible to perfuse trough the heart because during this operation some drugs, hormones or other are been release into the circulation and could induce apoptosis on my cells. I knew two of the papers taht Moleculardude suggested me, and in both cases they did vein perfusion. So I'm moving in this direction. Hope to be able.......
Anyway thank you a lot for your help, and if you have more suggestions, don't hesitate....
With best regards
This is my protocol:
1. Worm the collagenase type I 0.05% (Sigma, add the collagenase immediately before the use to a Buffer with HEPES, NaCl, KCl and KH2PO4, as reported in the paper of Seglen: Seglen PO. Preparation of isolated rat liver cells.Methods Cell Biol. 1976;13:29-83.) and another buffer with the same composition but w/o EGTA and with calcium.
2. Flow trough the pump until the pipes are completely filled of buffer A. Take 10 ml of Buffer for the collagenase digestion but w/o collagenase (separation buffer) and put into one Petri dish.
3. kill and fix the animal on the appropriate table:
• After washing with ethanol (70%, to avoid mycoplasma and hairs contaminations) the ventral of the mouse, make a small cut at the level of the pubis, such as to allow the entrance of one point of the scissors.
• Perform a median longitudinal cut superiorly to the chin, having cared to carefully remove the skin and the underlying musculature.
• Open with scissors the left thoracic cavity starting from the last rib by cutting the costal cartilages at their point of union with the bone. Continue cutting, until uncovering the pumping heart.
• Put the catheter (25 GA 0.50 In) of the pump into the heart (left ventricle), start to perfuse with washer Buffer (with EGTA) and at the same time cut the right atrium; when the liver became white (2-3 min) stop the pump and put the tube of the pump into a falcon 50 ml containing the collagenase type I in order to avoid any bubble formation into the tube. Switch on the pump continuing to perfuse for 5 min or more until the liver softening of the tissue gradually appears as well as marbling, finally the liver tissue appears like as sponge (as marker prick the liver and note the formation of white spot and the consistence of the tissue), consequently you have to regulate the speed of the pump in order to digest the liver very well. When perfusing put the catheter into the left ventricle (the left ventricle is under the right ventricle when the mouse is prone on the back; so is need to raise just a little the heart to start the perfusion) and cut the right atrium in order to flow the blood from the animal. Remove all the liver and put it quickly into the Petri dish containing separation buffer. Then cut it in small pieces with a scissors at room temperature. During this phase the pieces of liver release small particles (cluster oh hepatocytes)
4. Place the dish on ice to arrest the collagenase digestion.
5. By using a plastic pipette (Falcon, 3 ml) aspirate the media and filter it trough the mesh filter (50 micron), add 5-6 ml of separation buffer to the dish and aspirate and inject the pieces of liver very gently. Avoid aspirating and injecting biggest pieces of liver in order to not stress the cells very much. Repeat the procedure 4-5 times. If more than one mouse is sacrificed, put the tube containing the cell into the incubator.
6. Centrifuge the cells at 50 g for 2 min at room temperature.
7. Aspirate the separation Buffer and wash the pellet with the same buffer and repeat the centrifugation (this step is to be sure about the elimination of collagenase from the cells).
8. Discard the supernatant and dilute with fresh medium the cells in an appropriate volume.
9. After leave the cells to attach to the bottom for 4 hours or 1 day and then replace the medium. Sometimes when replace the medium we can detach the cells, so another procedure is to add fresh medium without removing the old medium, bur in this case be warning to the amount of medium into the well.