TITLE: Mammalian cell culture and transfection for stable cell lines generation
AUTHORS: Hina Ojha, Miratul M. K. Muqit

[DESCRIPTION]
Autosomal recessive mutations in PTEN-induced kinase 1 (PINK1) are linked to early-onset Parkinson's disease (PD) [1]. Upon mitochondrial depolarization, PINK1 activates through autophosphorylation and stabilization on mitochonria [2]. Pink1 phosphorylates ubiquitin and Parkin, triggering mitophagy to remove damaged mitochondria in PD [3]. To delve deeper into the impact of PINK1 mutations, a PINK1 knockout (KO) HeLa cell line was utilized as a model system. Additionally, stable cell lines with mutated PINK1 were established to explore differences in functional activity and the formation of the PINK1-TOM complex between wild-type PINK1 and its mutant variants.

[STEPS]
SECTION: Cell Culture
1. Maintain cells at 37 °C in a 5% CO2 water-saturated incubator.
SECTION: Generation of Stable Cell Lines:
6. Achieve doxycycline-induced, stable expression of exogenous protein using the Flp-In T-Rex system according to Invitrogen's instructions, utilizing CRISPR knock-out PINK1 KO HeLa Flp-In T-Rex cells [4]. The exact steps are detailed below.
SECTION: Cell Culture
3. The cell culture passages usually used are from P10 to P20. The passages are never used above P25.
SECTION: Cell Culture
2. Grow HeLa cells in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS), 2mM L-glutamine, 100 U/ml penicillin, and 0.1 mg/ml streptomycin (complete media).
SECTION: Maintenance of HeLa Flp-In T-Rex Stable Cell Lines:
4. For HeLa Flp-In T-Rex stable cell lines, use complete media supplemented with blasticidin and zeocin before recombination/transfection for stable cell line generation.
SECTION: Maintenance of HeLa Flp-In T-Rex Stable Cell Lines:
5. Supplement with blasticidin and hygromycin B following recombination/transfection.
SECTION: Generation of Stable Cell Lines:
7. Maintain HeLa PINK1 knock-out Flp-In T-Rex cells in blasticidin and zeocin.
SECTION: Generation of Stable Cell Lines:
8. Wash cells with PBS wash and switch to complete media 1440 min before transfection. 

SECTION: Generation of Stable Cell Lines:
9. Carry out transfection by co-transfecting 0.5 µg integratable hygromycin-resistant pcDNA FRT/TO vector of desired PINK1/mutant with 4.5 µg pOG44 expressing the Flp recombinase using Lipofectamine3000 in 100mm Petri dish [5, 6].

 
 
SECTION: Generation of Stable Cell Lines:
10. Mix the 2 tubes and keep at RT for 15 min.
SECTION: Generation of Stable Cell Lines:
11. Add the transfection mix drop by drop in the plate containing HeLa PINK1 knock-out Flp-In T-Rex cells. Keep a plate of untransfected cells as a negative control.
SECTION: Generation of Stable Cell Lines:
12. After 2880 min of transfection, split the cells with around 25% confluency. 
SECTION: Generation of Stable Cell Lines:
13. Once the cells are attached, add fresh complete media supplemented with blasticidin and hygromycin.
SECTION: Generation of Stable Cell Lines:
14. Maintain the cells with regular media changes every 2-3 days. Remove dying/dead cells when required. If successful, you will see separate colonies growing. Colonies amount varies from 10-50 per plate.

SECTION: Generation of Stable Cell Lines:
15. Trypsinize surviving colonies after 3-4 weeks of selection.
SECTION: Generation of Stable Cell Lines:
16. Expand the selected colonies, and induce protein expression with 0.02 μM doxycycline.
SECTION: Treatment with Mitochondrial Uncoupler:
17. Prepare a 50 mM stock of Antimycin and 6.3 mM of Oligomycin in DMSO, and store at -20 °C.
SECTION: Treatment with Mitochondrial Uncoupler:
18. Uncouple mitochondria by treating with 10 μM of Antimycin A and 1 μM of  of Oligomycin for 180 min-360 min, using an equivalent volume of DMSO for control conditions.

SECTION: Cell Lysis and Mitochondrial Enrichment:
19. Whole cell lysis
SECTION: Cell Lysis and Mitochondrial Enrichment:
19.1. For collection keep plates with cells on ice covered with aluminium foil to provide even cool surface.
SECTION: Cell Lysis and Mitochondrial Enrichment:
19.2. Wash the cells with PBS and collect the cells with cell scraper.
SECTION: Cell Lysis and Mitochondrial Enrichment:
19.3. Collect the cells by centrifugation at 800 x g, 5 min, 4 °C.
SECTION: Cell Lysis and Mitochondrial Enrichment:
19.4. Add around 300 µL of Lysis buffer for 100 mm cell plate lysate. Resuspend the cells with lysis buffer containing 1% triton and keep them on ice for 30 min.
SECTION: Cell Lysis and Mitochondrial Enrichment:
19.5. Clarify lysates by centrifugation at 17000 x g, 20 min, 4 °C.

SECTION: Mitochondrial Enrichment:
20. For collection keep plates with cells on ice covered with aluminium foil to provide even cool surface.
SECTION: Mitochondrial Enrichment:
21. Wash the cells with PBS and collect the cells with cell scraper.
SECTION: Mitochondrial Enrichment:
22. Collect the cells by centrifugation at 800 x g, 5 min, 4 °C.
SECTION: Mitochondrial Enrichment:
23. Pellet down the cells at 800 x g, 5 min, 4 °C. For 150 mm plate cell pellet add 300 µL of mitochondria fractionation buffer.
SECTION: Mitochondrial Enrichment:
24. Disrupt cell membranes using a 25-gauge needle by passing through it for 25 times on ice.

SECTION: Mitochondrial Enrichment:
25. Clarify lysates by centrifugation at 800 x g, 10 min, 4 °C.

SECTION: Mitochondrial Enrichment:
26. Discard the cytoplasmic membrane/nucleus/debris pellet.
SECTION: Mitochondrial Enrichment:
27. Isolate supernatant and centrifuge at 17000 x g, 20 min, 4 °C to collect mitochondrial enriched fraction.
SECTION: Mitochondrial Enrichment:
28. Keep supernatant as the cytoplasmic fraction.
SECTION: Mitochondrial Enrichment:
29. Snap-freeze the mitochondrial enriched pellet for Blue native PAGE or resuspend the pellet in mitochondria fractionation buffer with 1% Triton X-100 to keep as the mitochondrial-enriched fraction.