Aug 07, 2024
Version 2
URMC TriState SenNet Mouse Lung Digestion V.2
Version 1 is forked from Mouse Ischemia Experiment
- 1Department of Environmental Medicine, University of Rochester Medical Center, Rochester, NY;
- 2University of Rochester
- TriState SenNet
Protocol Citation: Gagandeep Kaur, Irfan Rahman 2024. URMC TriState SenNet Mouse Lung Digestion. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvok9z9l4o/v2Version created by Gagandeep Kaur
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 07, 2024
Last Modified: August 07, 2024
Protocol Integer ID: 104916
Keywords: cellular senescence, lung tissue, Dissociation, urmc tristate sennet mouse lung digestion the objective, urmc tristate sennet mouse lung digestion, tristate sennet tmc bioanalyses core, tristate sennet tmc bioanalyses core at the university, scrna analysis, cellular senescence, part of the cellular senescence network program, mouse lung, cellular senescence network program, minimum damage to the cell
Funders Acknowledgements:
TriState SenNet
Grant ID: U54AG075931
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.
Materials
Materials, Equipment & Reagents including Formulations
- Liberase: (Cat# 5401127001; Roche)
- DNase I (Cat# 4380-096-06; R&D)
3. Serum free DMEM
4. Disposables:
i) C-tubes for GentleMACS (Cat# 130-093-237, Miltenyi Biotec)
ii) MACS SmartStrainers 70µm (Cat# 130-098-462, Miltenyi Biotec)
iii) Sterile serological pipets; 50, 25,10, 5, 2, 1 mL size
iv) Sterile pipet tips; 1000, 200 and 20 μl
v) Sterile 15mL or 50mL conical polypropylene tubes
vi) Test tube racks for 15mL or 50mL conical tubes
vii) LD columns (Cat# 130-042-901; Miltenyi Biotec)
5. Equipment:
i) GentleMACS Tissue Octo Dissociator (Miltenyi Biotec Inc San Diego, CA)
ii) MACSmix tube rotator
iii) Table Top Centrifuge
iv) Biological Safety Cabinet, Class II
v) Dissecting forceps and scalpel
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