Mouse Ischemia Experiment

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)

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

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.

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.

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).

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.

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.

3. Centrifuge the cells at 300 g for 5 min and wash 2x with FACS buffer.

4. Discard the supernatant by inversion and resuspend the cell pellet in the remain volume (≈ 100 ul).

5. Perform a co-immunolabeling to further characterize the population of interest.

PS: from this point keep the samples out of direct light.

1. Prepare the blocking reagent by making a 1:10 dilution (1µL in 100µL of PBS) of CD16/32. Incubate at 4⁰C for 10 minutes.

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.

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.

4. Master Mix for Samples (markers change depending upon the experiment):

A	B	C
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

5. Vortex gently & incubate @ 4°C (place in frig) for 20 minutes.

6. Wash with 1ml 1xPBS & centrifuge @ 500g for 5 min @ 4 degrees

7. Repeat 1xPBS wash a second time & centrifuge @ 500g for 5min @ 4 degrees.

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.