Apr 30, 2026
Culturing, transfection and NBD-labeled lipid uptake assay in HeLa-CDC50A-OE cells
- 1Aarhus University
Protocol Citation: Klara Scholtissek, Filip Pamula 2026. Culturing, transfection and NBD-labeled lipid uptake assay in HeLa-CDC50A-OE cells . protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6zo8kgqe/v1
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 10, 2025
Last Modified: April 30, 2026
Protocol Integer ID: 234648
Keywords: ASAPCRN, lipid uptake assay in hela, labeled lipid uptake assay, lipid uptake measurement, oe cell, flow cytometry, transient overexpression of flippase construct
Funders Acknowledgements:
ASAP
Grant ID: ASAP-024297
Abstract
The NBD‑labeled lipid uptake assay outlines the procedures for seeding, transient transfection, and lipid uptake measurement in HeLa‑CDC50A‑OE cells, monitored by flow cytometry. This assay enables transient overexpression of flippase constructs and allows quantitative assessment of their NBD‑lipid uptake activity within a cellular environment.
Culturing HeLa-CDC50A-OE cells
HeLa-CDC50A-OE cells are kept at 37°C and 5% CO2 in a humidified incubator in DMEM medium supplemented with 10% FBS, 1% P/S and for selection 160µg/ml hygromycin. They require passaging 1:10 two times a week.
Cell seeding & transfection
Seed 90,000 HeLa-CDC50A-OE cells per one well of a 12-well plate
Cell seeding & transfection
Remove DMEM from adherent cells
Wash cells with PBS
Add Trypsin to detach cells, incubate at 37°C for 5 minutes
Neutralize Trypsin by addition of DMEM supplemented with 10% FBS and 1 % Pen-Strep
Count cells and determine viability
Dilute cells to 90,000 cells/ml in DMEM
Pipette 1 ml of cell suspension per one well in a 12-well plate
Rock back and forth immediately
Incubate at 37°C and 5 % CO2 for 16-24 hrs
Transfect HeLa-CDC50A cells with PEI
For a single well: incubate 55 µl of serum free DMEM with 1 µg of DNA and in a second eppendorf tube incubate 55 µl of serum free DMEM with 5 µg PEI for 15 minutes at RT. Vortex thoroughly after every pipetting step
Mix the PEI mixture into the DNA mixture. Vortex thoroughly. Incubate 15 minutes at RT
Remove DMEM from seeded cells in 12-well plate
Wash with 1 ml PBS
Replace PBS with 300 µl serum free DMEM
Drop transfection mixture onto the cells while shaking gently
Incubate cells overnight at 37°C and 5 % CO2
Monitor expression by fluorescence if available
Replace transfection mixture with fresh DMEM supplemented with 10% FBS and 1 % Pen-Strep
Lipid uptake assay
Remove DMEM from cells
Wash with 1 ml PBS
Cover with 460 µl HBSS
Equilibrate cells for 20 minutes to 15°C
Replace HBSS with 1 µM NBD-labeled lipid in HBSS (pre-equilibrated to 15°C)
Incubate for 20 minutes at 15°C
Replace lipid solution by 2.5 % BSA in HBSS with 15 mM EDTA and keep cells from here on on ice
Detach cells by pipetting of the plate
Filter through filter mesh into flow cytometry tube
Add DRAQ7 to a final concentration of 3 µM, vortex cells gently
Analysis by Flow Cytometry
Cells are analyzed on a NovoCyte Quanteon 4025 flow cytometer equipped with four lasers (405 nm,
488 nm, 561 nm and 637 nm) and 25 fluorescence detectors (Agilent, Santa Clara,
CA) provided by FACS core facility at Århus Universitet run with Software: NovoExpress (v. 1.6.2, Agilent, Santa Clara, CA)
After excluding dead cells based on DRAQ7 staining, 2,000 - 10,000 events were analyzed in either mCherry-positive or expression positive quadrant gate
Lasers and filters used:
For BFP-detection: 405 nm laser Violet - Filter: V445 - laser gain adjusted to 500
For NBD-detection: 488 nm laser Blue - Filter: B530
For mCherry-detection: 561 nm laser YellowGreen - Filter: Y615
For DRAQ7-detection: 637 nm laser - Red - Filter: R695
Normalize expression by dividing the median NBD-A by the median mCherry-A of the respective population