Dear all,
I would like to purify lung mononuclear cells from BALB/c mouse and detect CD4+ T cells by FACS. At first, I tried to isolate cell suspension by collagenase digestion and to proceed to cell labeling. However, there were too many cell deaths as well as changes in cell morphology.
Another protocol suggested to isolate the cell suspension simply by scissor, followed by density gradient centrifugation with Ficoll-Hypaque to purify lung mononuclear cells from rat lung. Is it possible to conduct this step? Beside, could you please recommend me any possible protocols?
Thank you
how did you determine cell viability? if you used trypan blue exclusion, contaminating RBCs will be counted as stained cells on automated cell counters.
Dear Tu,
Attached please find a publication that covers the answer to your question. I have copied important paragraph from the Methods section for quick view.
Isolation of Lung Resident Mononuclear Cells. Lungs were harvested after
perfusion with PBS. Lungs were first inflated with 0.1 mg/mL collagenase IV
and DNase1 for 15 min at 37 °C. Single cell suspensions were prepared by
mechanical dissociation of lung tissue through a 70-μm nylon mesh. Lung cells
were suspended in PBS and layered on LSM Lymphocyte Separation (Medium
MP Biomedical). Cells were centrifuged at room temperature for 20 min at
900 × g. Mononuclear cells were harvested from the gradient interphase.
Hoping this will be helpful,
Rafik
Dear Tu! I am trying to isolate also lung mononuclear cells from mice, without success... Can you share the protocol you are using? Thank you in advance!
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