TITLE: Mouse Ischemia Experiment
AUTHORS: gagandeep kaur, Irfan_Rahman

[DESCRIPTION]
The objective of this protocol is to understand the effect of cold ischemia time on cellular senescence in mouse lung and heart tissues.

The sampling of lung and heart tissues will be done based on equal weights. 6 pieces weighing around 40-50mg will be cut and put in cold preservative to conduct the time-controlled experiment.

As a positive control for C12FDG a technical control of cells treated with EtOH (500µM) will be used. 

[STEPS]
SECTION: Lung Tissue Dissociation
2. 
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.

SECTION: Lung Tissue Dissociation
1. 1)    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)
SECTION: Lung Tissue Dissociation
3. 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.
SECTION: Lung Tissue Dissociation
4. Remove conical from rocker and strain the cell suspension through 70 micron into 50mL tube 
SECTION: Lung Tissue Dissociation
5. Add 3 ml DMEM (10% FBS) through the strainer to collect the remaining cell suspension
SECTION: Lung Tissue Dissociation
6. After straining, centrifuge at 300g for 5 minutes at 4degree C.
SECTION: Lung Tissue Dissociation
7. Remove supernatant, add 500 µl of red blood cell lysis to cell pellet. Incubate the cell suspension on ice for 5 min.
SECTION: Lung Tissue Dissociation
8. After incubation add 4.5 mL of DMEM (10% FBS) to the suspension and centrifuge at 300g for 5 minutes at 4degree C.
SECTION: Lung Tissue Dissociation
9. Remove supernatant, re-suspend pellet in 2 mL DMEM (10%FBS)
SECTION: Lung Tissue Dissociation
10. Count cells and viability using AO/PI method, confirm reading under microscope
SECTION: Treatment for positive control
11. After cell counting, divide the cell fractions into three equal parts. For each sample take 1 million cell and pool the remaining cells to make a heterogeneous cell population. Divide this heterogeneous cell population into three fractions. One for positive control and other as negative control and one as a single stained control for C12FDG. Make up the final volume in each tube as 5mL.
SECTION: Treatment for positive control
12. For positive controls, treat the cell fraction with 500 µM EtOH, and leave the rest of the two fractions as is. EtOH stock = 17.3M; For treatment first make a first dilution of 500mM EtOH in 1 mL media (29.4 µL in 970 µl of DMEM). Add 5 uL of the first dilution (500mM) to 5mL volume of cell suspension in positive control tube to make the final concentration of 500µM.
SECTION: Treatment for positive control
13. Incubate all the tubes in CO2 incubator for 2 hrs. (NOTE: All this could be done in a 15 mL tube. Make sure the tube caps are loosened when placed in the incubator).
SECTION: Measure of senescence (C12FDG) by flow cytometry
14. C12FDG (FITC channel):

1. Treat the cells with a final concentration of 100 nM of bafilomycin A1. For this add 5µL of 0.1 mM dilution to each tube with 5mL cell suspension. Incubate 1 h at 37°C, 5% CO2 
PS: Bafilomycin A must be added to the negative control tube as well.
SECTION: Measure of senescence (C12FDG) by flow cytometry
15. 2. Add C12FDG to cells at a final concentration of 20 uM. For this add 50uL of 2mM of C12FDG stock solution to each 5mL cell suspension tube (except negative control). Incubate for 1.5 h at 37°C, 5% CO2. Leave one fraction of cells untreated. This will act as our negative control for C12FDG treatment.
SECTION: Measure of senescence (C12FDG) by flow cytometry
16. 3. Centrifuge the cells at 300 g for 5 min and wash 2x with FACS buffer.
SECTION: Measure of senescence (C12FDG) by flow cytometry
17. 4. Discard the supernatant by inversion and resuspend the cell pellet in the remain volume (≈ 100 ul).
SECTION: Measure of senescence (C12FDG) by flow cytometry
18. 5. Perform a co-immunolabeling to further characterize the population of interest. 
SECTION: Co-immunolabelling
19. PS: from this point keep the samples out of direct light.
Prepare the blocking reagent by making a 1:10 dilution (1µL in 100µL of PBS) of CD16/32. Incubate at 40C for 10 minutes. 
SECTION: Co-immunolabelling
20. 2. Thereafter add 10 µL of the blocking buffer to each sample tube, vortex and incubate at 40C for 10 minutes
Following incubation, centrifuge the cells at 500g for 5 min at 40C and wash once with 1 mL PBS. Finally, resuspend in 75uL of FACS buffer. Add 25ul of the 75ul of each sample to the unstained tube.
SECTION: Co-immunolabelling
21. 3. Create bead and single stained cell controls. For bead controls one drop beads with 1µL of  each antibody to make single stained controls. DO NOT forget to make unstained bead controls. 
Likewise, can make for cells suspension as well.
SECTION: Co-immunolabelling
22. 4. Master Mix for Samples (markers change depending upon the experiment):

 
 
 
 
     Antibody      Dilution      Volumes   to be added in 1mL PBS        CD326 (APC-Cy7)       1:1000      1 µL        CD140a (PE)      1:750      1.5 µL        CD31 (APC)      1:1000      1 µL        CD26/DPP4      1:750      1.5 µl   

Add 50ul to control and sample tubes. Do not add any master mix to unstained control tubes or C12FDG only tube.
PS: Do not add the antibodies to the single stained tube for C12FDG
 
 
 
 
SECTION: Co-immunolabelling
23. 5. Vortex gently & incubate @ 4°C (place in frig) for 20 minutes.
SECTION: Co-immunolabelling
24. 6. Wash with 1ml 1xPBS & centrifuge @ 500g for 5 min @ 4 degrees
SECTION: Co-immunolabelling
25. 7. Repeat 1xPBS wash a second time & centrifuge @ 500g for 5min @ 4 degrees.
SECTION: Co-immunolabelling
26. 8. Resuspend in 150ul 1x FACs buffer and run samples on flow-cytometer. 

The Capture of the cells must be done at both 10000 and 100,000 counts for gating.