TITLE: Mitochondrial isolation from HeLa cells
AUTHORS: Louise Uoselis

[DESCRIPTION]
Mitochondrial isolation and quantification from HeLa cells.

[STEPS]
SECTION: Mitochondrial isolation
1.      Thaw cell pellets that have been frozen at -80 °C for at least 15 min, on ice.

SECTION: Mitochondrial isolation
2. Aliquot out an amount of Solution B (220 millimolar (mM) mannitol, 70 millimolar (mM) sucrose, 1 millimolar (mM) EDTA, 20 millimolar (mM) HEPES-KOH pH 7.6) assuming for5 mL of Solution B per sample (if samples have been collected from 10cm plates), and add PMSF at a final concentration of 0.5 millimolar (mM).  Keep the Solution B on ice at all times. 

SECTION: Mitochondrial isolation
3. Resuspend the first sample in 3 mL of Solution B (+PMSF), and homogenize with 30 strokes using a Dounce hand-held glass homogenizer (7 mL capacity)

SECTION: Mitochondrial isolation
4. Move the homogenate back to the original tube, and wash the homogenizer using distilled water. 
SECTION: Mitochondrial isolation
5. Repeat steps 3 – 4 for each sample. 
SECTION: Mitochondrial isolation
6. After all samples have been homogenised, spin all samples for 5 min at 4 °C and 800 rcf .

SECTION: Mitochondrial isolation
7. Transfer the supernatant to microfuge tubes (will need 3 microfuge tubes per sample if starting with 3 mL), being careful to not touch the pellet. 
SECTION: Mitochondrial isolation
8. Spin the supernatant for 10 min at 4 °C and 10000 rcf. The resulting pellet will contain mitochondria. 

SECTION: Mitochondrial isolation
9. Carefully aspirate the supernatant from each tube.
SECTION: Mitochondrial isolation
10. Collate the individual tubes for each sample into one microfuge tube, by resuspending mitochondrial pellets in 200 µL of Solution B and pooling together into one tube. Rinse each tube with another 200 µL of Solution B, and combine these two volumes together into the one tube. 

SECTION: Mitochondrial isolation
11. Spin the resuspended mitochondria for 10 min at 4 °C and 10000 rcf.

SECTION: Mitochondrial isolation
12. Carefully aspirate the supernatant from each tube. 
SECTION: Mitochondrial isolation
13. Resuspend each sample in ~300-400 uL of Sucrose Storage Buffer (0.5 Molarity (M) sucrose, 10 millimolar (mM) HEPES-KOH pH 7.6) (depending on the size of the mitochondrial pellet). Aliquot 15 µL into a separate tube (to be used for protein quantification), and then split the sample into three separate tubes for storage (this is to allow the samples to be used more times without being freeze-thawed excessively). Store mitochondria in Sucrose Storage Buffer at -80 °C. When required, thaw the samples on ice before use. 

SECTION: Quantification of mitochondrial samples
14. Centrifuge the 15 µL aliquot taken from each sample at 4 °C for 10 min at 10000 rcf. 

SECTION: Quantification of mitochondrial samples
15. Carefully aspirate the supernatant from each tube. 
SECTION: Quantification of mitochondrial samples
16. Add 5 µL of 1x LDS lysis buffer (ThermoFisher) to each sample. 

SECTION: Quantification of mitochondrial samples
17. Boil samples at 99 °C shaking at maximum speed for 10 min. 

SECTION: Quantification of mitochondrial samples
18. Let samples cool to Room temperature, quickly centrifuge to collate the liquid at the bottom of the tube, and vortex each sample to ensure it is homogenous. 

SECTION: Quantification of mitochondrial samples
19. Blanking with the 1x LDS lysis buffer, measure the protein level spectroscopically using an A280 measurement. 
SECTION: Quantification of mitochondrial samples
20. Divide each concentration by 3, and that will represent the concentration of mitochondria in each sample.