Mouse Lung Digestion
gagandeep kaur, Irfan Rahman
Disclaimer
The protocols.io team notes that research involving animals and humans must be conducted according to internationally-accepted standards and should always have prior approval from an Institutional Ethics Committee or Board.
Abstract
The objective of this protocol is to digest the mouse lungs to perform scRNA analyses
Steps
Lung Tissue Dissociation
- Mince tissue finely, place into 50ml GentleMACS C-tube with liberase enzymatic cocktail a. 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 and m_heart_01.01 for mouse heart.
Thereafter, place 50ml conical with digestion into an incubating rocker for 30 minutes at 37 degrees 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 4degree 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 4degree C.
Remove supernatant, re-suspend pellet in 2 mL DMEM (10%FBS)
Count cells and viability using AO/PI method, confirm reading under microscope
Submitted the digested suspension to the Genomic Research Center for scRNA seq analyses
