Mammalian cell culture and transfection for stable cell lines generation
Miratul M. K. Muqit, Hina Ojha
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Abstract
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.
Attachments
Steps
Cell Culture
Maintain cells at 37°C in a 5% CO2 water-saturated incubator.
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).
The cell culture passages usually used are from P10 to P20. The passages are never used above P25.
Maintenance of HeLa Flp-In T-Rex Stable Cell Lines:
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.
Supplement with blasticidin and hygromycin B following recombination/transfection.
Generation of Stable Cell Lines:
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.
Maintain HeLa PINK1 knock-out Flp-In T-Rex cells in blasticidin and zeocin.
Wash cells with PBS wash and switch to complete media 24h 0m 0s before transfection.
Carry out transfection by co-transfecting 0.5µg integratable hygromycin-resistant pcDNA FRT/TO vector of desired PINK1/mutant with 4.5µg pOG44 expressing the Flp recombinase using Lipofectamine3000 in 100mm Petri dish [5, 6].
| A | B | C | D |
|---|---|---|---|
| A | B | C | D |
| Tube 1 | **** | **** | **** |
| POG44 plasmid | 4.5 µg | ||
| Desired DNA plasmid | 0.5 µg | Total DNA = 5µg | |
| Lipofectamine P3000 reagent | 10 µl | ||
| Opti-MEM | 0.5 ml | ||
| Tube 2 | |||
| Lipofectamine reagent | 7.5 µl | ||
| Opti-MEM | 0.5 µl |
Mix the 2 tubes and keep at RT for 0h 15m 0s.
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.
After 48h 0m 0s of transfection, split the cells with around 25% confluency.
Once the cells are attached, add fresh complete media supplemented with blasticidin and hygromycin.
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.
Trypsinize surviving colonies after 3-4 weeks of selection.
Expand the selected colonies, and induce protein expression with 0.02μM doxycycline.
Treatment with Mitochondrial Uncoupler:
Prepare a 50mM stock of Antimycin and 6.3mM of Oligomycin in DMSO, and store at -20°C.
Uncouple mitochondria by treating with 10μM of Antimycin A and 1μM of of Oligomycin for 3h 0m 0s-6h 0m 0s, using an equivalent volume of DMSO for control conditions.
Cell Lysis and Mitochondrial Enrichment:
Whole cell lysis
For collection keep plates with cells On ice covered with aluminium foil to provide even cool surface.
Wash the cells with PBS and collect the cells with cell scraper.
Collect the cells by centrifugation at 800x g,4°C.
Add around 300µL of Lysis buffer for 100mm cell plate lysate. Resuspend the cells with lysis buffer containing 1% triton and keep them On ice for 0h 30m 0s.
Clarify lysates by centrifugation at 17000x g,4°C.
Mitochondrial Enrichment:
For collection keep plates with cells On ice covered with aluminium foil to provide even cool surface.
Wash the cells with PBS and collect the cells with cell scraper.
Collect the cells by centrifugation at 800x g,4°C.
Pellet down the cells at 800x g,4°C. For 150 mm plate cell pellet add 300µL of mitochondria fractionation buffer.
Disrupt cell membranes using a 25-gauge needle by passing through it for 25 times On ice.
Clarify lysates by centrifugation at 800x g,4°C.
Discard the cytoplasmic membrane/nucleus/debris pellet.
Isolate supernatant and centrifuge at 17000x g,4°C to collect mitochondrial enriched fraction.
Keep supernatant as the cytoplasmic fraction.
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.
