Dec 13, 2023
Version 1
Cell culture, transfection, and imaging of K562 cells V.1
- 11Departments of Neuroscience and of Cell Biology, Howard Hughes Medical Institute, Program in Cellular Neuroscience, Neurodegeneration and Repair, Yale University School of Medicine, New Haven, Connecticut 06510, USA 2Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815
Protocol Citation: Chase Amos, Pietro De Camilli 2023. Cell culture, transfection, and imaging of K562 cells. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwdk4dlmk/v1Version created by Chase Amos
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 13, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 92286
Keywords: ASAPCRN, imaging of k562 cell, general preparation of k562 cell, k562 cell, cell culture, cell, imaging, transfection
Abstract
This protocol details the general preparation of K562 cells for imaging.
Cell culture
2d 17h 30m
Culture K562 cells at 37 °C and 5% CO2 in RPMI containing 10% FBS, 1 millimolar (mM) sodium pyruvate, 100 Mass Percent penicillin, 100 Mass Percent streptomycin, 2 millimolar (mM) L-glutamine, 1 Mass Percent non-essential amino acids, (all from Gibco) and 2.5 Mass Percent plasmocin (InvivoGen).
Note
K562 cells are maintained between 1x105 and 1x106 cells/mL, passaging when reaching confluency (1x106 cells/ml) or otherwise renewing media every 2-3 days.
Transfection and imaging
2d 17h 30m
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 Overnight at 37 °C and 5% CO2.
16h
After cells attach, replace media with RPMI supplemented with hemin (Sigma Aldrich) dissolved in DMSO to a final concentration of 30 micromolar (µM) . Transfect transiently with plasmids by adding FuGene 4K (Promega) and incubate Overnight . 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 Overnight .
2d
Following the 2 days of transfection/hemin treatment, prepare the cells for imaging. Where applicable, add Halo ligand and incubate for 01:30:00 at 37 °C , 5% CO2. Replace with new RPMI media (supplemented at 30 micromolar (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 200 nanomolar (nM) TMRE.
1h 30m
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.