Cell culture, transfection, and imaging of K562 cells
Pietro De Camilli, Chase Amos
Abstract
This protocol details the general preparation of K562 cells for imaging.
Steps
Cell culture
Culture K562 cells at 37°C and 5% CO2 in RPMI containing 10% FBS, 1millimolar (mM) sodium pyruvate, 100 penicillin, 100 streptomycin, 2millimolar (mM) L-glutamine, 1 non-essential amino acids, (all from Gibco) and 2.5 plasmocin (InvivoGen).
Transfection and imaging
For imaging experiments, seed the cells on fibronectin (Sigma Aldrich) 35mm imaging dishes (MatTek) at a concentration of 2x105 cells per dish in RPMI without antibiotics. Allow cells to attach 16h 0m 0s at 37°C and 5% CO2.
After cells attach, replace media with RPMI supplemented with hemin (Sigma Aldrich) dissolved in DMSO to a final concentration of 30micromolar (µM). Transfect transiently with plasmids by adding FuGene 4K (Promega) and incubate . After the overnight incubation, replace the media with new media containing the same factors of hemin and (where applicable) transfection reagent and plasmids, and incubate .
Following the 2 days of transfection/hemin treatment, prepare the cells for imaging. Where applicable, add Halo ligand and incubate for 1h 30m 0s at 37°C , 5% CO2. Replace with new RPMI media (supplemented at 30micromolar (µM) hemin if differentiating cells) prior to imaging.
If using TMRE (Cayman Chemical, TMRE Mitochondrial Membrane Potential Assay Kit), add prior to imaging at a final concentration of 200nanomolar (nM) TMRE.
Perform spinning-disk confocal microscopy using an Andor Dragonfly system equipped with a plan apochromat objective (63×, 1.4 NA, oil) and a Zyla scientific CMOS camera. Cells are imaged at 37°C and 5% CO2.
Identify the cells to be imaged by scanning the dish.
