URMC TriState SenNet Mouse Lung Digestion
Irfan Rahman, Gagandeep Kaur
Disclaimer
The authors have no conflict of interest to declare.
Abstract
The objective of this document is to share the material and kits used and steps involved in digesting the mouse lungs to perform scRNA analyses. This study was performed for the TriState SenNet TMC Bioanalyses Core at the University of Rochester, as part of the Cellular Senescence Network Program (SenNet). This protocol ensures minimum damage to the cells in suspension and ensures maximum viability to conduct downstream analyses.
Steps
Lung Tissue Dissociation
Mince tissue finely, place into 50ml GentleMACS C-tube with liberase enzymatic cocktail .
For 50mg tissue use 0.5 ml liberase + 2ml DMEM + 2ul 5 unit/ul DNase I.
Immediately transfer the tubes to MACS Tissue Dissociator and run user-defined program named m_lung_01_02 for mouse lungs.
Thereafter, place 50ml conical with digestion into an incubating rocker for 30 minutes at 37°C .
NOTE: If you still see chunks of lung tissue in the tubes after this step then transfer to the MACS Tissue Dissociator and run user-defined program named m_lung_02_01 for mouse lungs for 10-15 sec.
Remove conical from rocker and strain the cell suspension through 70 micron into 50mL tube
Add 3 ml DMEM (10% FBS) through the strainer to collect the remaining cell suspension
After straining, centrifuge at 300g for 5 minutes at 4°C.
Remove supernatant, add 500 µl of red blood cell lysis to cell pellet. Incubate the cell suspension On ice for 5 min.
After incubation add 4.5 mL of DMEM (10% FBS) to the suspension and centrifuge at 300g for 5 minutes at 4°C .
Remove supernatant, re-suspend pellet in 2 mL DMEM (10%FBS)
Count cells and viability using AO/PI method, confirm reading under microscope
