Jul 28, 2023

Public workspaceCellular lipid uptake with flow cytometry readout

DOI
https://dx.doi.org/10.17504/protocols.io.14egn2pnpg5d/v1
  • Igor Beletchi1,
  • rosanne.wouters 1,
  • Peter Vangheluwe1
  • 1KU Leuven
rosanne.wouters
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DOI: https://dx.doi.org/10.17504/protocols.io.14egn2pnpg5d/v1
Protocol Citation: Igor Beletchi, rosanne.wouters , Peter Vangheluwe 2023. Cellular lipid uptake with flow cytometry readout. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn2pnpg5d/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 31, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 82703
Keywords: ASAPCRN, cellular lipid uptake with flow cytometry, cellular lipid uptake, flow cytometry, lipid
Funders Acknowledgements:
ASAP (Aligning Science Across Parkinson's)
Abstract
Cellular lipid uptake with flow cytometry readout
Protocol materials
ReagentTrypLE Express or preferred cell dissociation reagentThermo Fisher Scientific
ReagentPBS without Ca2 or Mg2 Gibco - Thermo Fisher ScientificCatalog #10010-031
ReagentHBSSGibco - Thermo Fisher ScientificCatalog #14170-112
ReagentNBD-SM (C6 NBD sphingomyelin) Avanti Polar Lipids, Inc.Catalog #810218C
ReagentNBD-GluCer (C6-NBD Glucosyl Ceramide)Avanti Polar Lipids, Inc.Catalog #810222C
ReagentNBD-PC (18:1-06:0 NBD PC)Avanti Polar Lipids, Inc.Catalog #810132C
ReagentNBD-lyso-PC (12:0 lyso NBD PC) Avanti Polar Lipids, Inc.Catalog #810128C
ReagentNBD-PS (18:1-06:0 NBD PS) Avanti Polar Lipids, Inc.Catalog #810194C
ReagentNBD-PE (18:1-06:0 NBD PE) Avanti Polar Lipids, Inc.Catalog #8105155C
ReagentAlbumin Bovine Serum Fraction V Fatty Acid-FreeMerckCatalog #126575
Troubleshooting
harvest cells
10m
harvest cells by detachment with ReagentTrypLE Express or preferred cell dissociation reagentThermo Fisher Scientific

cells should be grown to 70% confluency, not higher
Critical
wash cells with ReagentTrypLE Express or preferred cell dissociation reagentThermo Fisher Scientific

incubate cells with ReagentTrypLE Express or preferred cell dissociation reagentThermo Fisher Scientific (3ml/T175 flask) at room temperature

when cells detach resuspend with 7ml ReagentPBS without Ca2 or Mg2 Gibco - Thermo Fisher ScientificCatalog #10010-031 and keep on ice in 15 mL falcon

collect cells by centrifugation Centrifigation400 x g, 4°C, 00:05:00

5m
Centrifigation
wash pellet with with ReagentPBS without Ca2 or Mg2 Gibco - Thermo Fisher ScientificCatalog #10010-031
collect cells by centrifugation Centrifigation400 x g, 4°C, 00:05:00
5m
resuspend pellet in ReagentHBSSGibco - Thermo Fisher ScientificCatalog #14170-112
count cells
prepare cell suspension with 1*10^6 cells in 500µl ReagentHBSSGibco - Thermo Fisher ScientificCatalog #14170-112

prepare lipid suspension
work under fume hood equipped with N2 flow
wash glass syringe 3x with 100% ethanol and 3x with chloroform, let dry
with glass syringe pipette needed volume of NBD-lipid into glass vial ReagentNBD-PC (18:1-06:0 NBD PC)Avanti Polar Lipids, Inc.Catalog #810132C ,ReagentNBD-lyso-PC (12:0 lyso NBD PC) Avanti Polar Lipids, Inc.Catalog #810128C ,ReagentNBD-PS (18:1-06:0 NBD PS) Avanti Polar Lipids, Inc.Catalog #810194C ,ReagentNBD-PE (18:1-06:0 NBD PE) Avanti Polar Lipids, Inc.Catalog #8105155C , ReagentNBD-SM (C6 NBD sphingomyelin) Avanti Polar Lipids, Inc.Catalog #810218C ,ReagentNBD-GluCer (C6-NBD Glucosyl Ceramide)Avanti Polar Lipids, Inc.Catalog #810222C

keep glass bottle with NBD-labeled lipids in chloroform solution cold, by working on ice or in freezer block
for each sample 500µl lipid suspension with 2µM final concentration is needed
dry lipids by evaporating chloroform under N2 flow
resuspend lipids in ReagentHBSSGibco - Thermo Fisher ScientificCatalog #14170-112 to final concentration of 2µM with magnetic stirbar until fully dissolved, keep lipids at 4°C while dissolving

lipid uptake
equilibrate cell suspension Shaker0 rpm, 37°C, 00:15:00
equilibrate lipid suspension Shaker0 rpm, 37°C, 00:15:00
prepare collection tubes with 200µl ReagentHBSSGibco - Thermo Fisher ScientificCatalog #14170-112 containing 5% ReagentAlbumin Bovine Serum Fraction V Fatty Acid-FreeMerckCatalog #126575 (per sample 1 collection tube is needed for each timepoint) and keep on ice

for timepoint 0, take 200µl of equilibrated cell suspension and add to collection tube T=0 (containing 200µl of ice-cold ReagentHBSSGibco - Thermo Fisher ScientificCatalog #14170-112 containing 5% ReagentAlbumin Bovine Serum Fraction V Fatty Acid-FreeMerckCatalog #126575 ) on ice
add 500µl of lipid suspension to remaining 300µl of cell suspension
incubate at 37°C with thermoshaker with interval 1 min shaking every 5 min (700 rpm).
keep samples away from light during incubation
after 30min, take 200µl of lipid+cell suspension and add to collection tubes T=30 (containing 200µl of ice-cold ReagentHBSSGibco - Thermo Fisher ScientificCatalog #14170-112 containing 5% ReagentAlbumin Bovine Serum Fraction V Fatty Acid-FreeMerckCatalog #126575 ), keep on ice
after 60min, take 200µl of lipid+cell suspension and add to collection tubes T=60 (containing 200µl of ice-cold ReagentHBSSGibco - Thermo Fisher ScientificCatalog #14170-112 containing 5% ReagentAlbumin Bovine Serum Fraction V Fatty Acid-FreeMerckCatalog #126575 ), keep on ice
add sodium dithionite to all samples (1/100 dilution of freshly prepared 1M stock in tris-buffer, pH10) and vortex samples (make sure to mix well with quencher)
measure total internal fluorescence with flow cytometer
Protocol references
The lipid uptake assay was adapted from (Naito et al., 2015).