May 11, 2026

Analysis of NBD-lipid uptake in transiently transfected HeLa-CDC50A cells by Flow Cytometry

DOI
https://dx.doi.org/10.17504/protocols.io.bp2l6jjkkvqe/v1
  • 1ASAP
Klara KTS Scholtissek
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DOI: https://dx.doi.org/10.17504/protocols.io.bp2l6jjkkvqe/v1
Protocol Citation: Klara Scholtissek 2026. Analysis of NBD-lipid uptake in transiently transfected HeLa-CDC50A cells by Flow Cytometry. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6jjkkvqe/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: May 11, 2026
Last Modified: May 11, 2026
Protocol  Integer ID: 316800
Keywords: ASAPCRN, cdc50a cells by flow cytometry, lipid uptake assay, flow cytometry, analysis by flow cytometry, labeled lipid uptake, cdc50a cell, lipid uptake, transfected hela, cell, analysis of nbd
Funders Acknowledgements:
ASAP
Grant ID: ASAP-024297
Abstract
The protocol describes the analysis of previously transiently transfected HeLa-CDC50A cells by flow cytometry. The cells were transfected with a flippase and conditions of interest and a lipid uptake assay was performed (see respective protocol). Here, the analysis by flow cytometry of NBD-labeled lipid uptake is described as well as data processing.
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)
Samples were maintained on a cooling block during analysis, and technical duplicates were measured sequentially which were averaged.
After excluding dead cells based on DRAQ7 staining, 2,000 - 10,000 events were analyzed in either mCherry-positive or respective 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
Export fcs files of all samples & import them into FlowJo.
Redraw following gates:
  1. on FSC vs SSC draw cell gate --> exclude cell debris in bottom left corner
  2. on FSC-A vs FSC-H draw singlet gate --> exclude doublets (below 45° line)
  3. on SSC-A vs DRAQ7-A draw live cell gate --> exclude DRAQ7 positive cells
  4. either draw NBD-A positive gate, mCherry-A positive gate or quadrant gates respectively
  5. export csv of the statistics including median of NBD-A, mCherry-A and BFP-A
Normalize expression by dividing the median NBD-A by the median mCherry-A of the respective population or if interested the median BFP-A by the median mCherry-A