I want to isolate monocytes and DC from liver. The protocol for density gradient cell separation I have now mentioned the speed for mononuclear cells (2500rmp, 20mins and all stuff should be room temperature) and for DC (1700g, 15mins and all stuff need to be cold).
However, I am a little confusing about: Do mononuclear cells include DC? If so, why we need to use different speeds and different temperature stuff? If not, is any possibility we isolate DC and monocytes from one liver? Coz liver looks bigger, but immune cells are not as much as we thought.
Did you applied collagenase perfusion before disintegration step?
Hi Petrenyov, when I cut liver into small pieces, I did apply collagenase into HBSS and incubation for 1hr
The best way to perfuse liver with collagenase solution - it could significantly increases viability and number of recovered cells.
Next step is gradient centrifugation (Percoll etc.) could be very stressful for cells (temperature, pH, O2 etc.). Depending of your aim it possible to collect Mf and ВС fraction by adhesion to plastic or use immunobeads depletion/enrichment.
- Badges
- Science topic
- Similar topics
- Biological Science
- Immunology
- Immunology Techniques