Cellular lipid uptake with flow cytometry readout

harvest cells by detachment with

cells should be grown to 70% confluency, not higher

wash cells with

incubate cells with (3ml/T175 flask) at room temperature

when cells detach resuspend with 7ml and keep on ice in 15 mL falcon

collect cells by centrifugation 400x g,4°C

wash pellet with with

collect cells by centrifugation 400x g,4°C

resuspend pellet in

count cells

prepare cell suspension with 1*10^6 cells in 500µl

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 , , , , ,

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 to final concentration of 2µM with magnetic stirbar until fully dissolved, keep lipids at 4°C while dissolving

equilibrate cell suspension rpm

equilibrate lipid suspension rpm

prepare collection tubes with 200µl containing 5% (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 containing 5% ) 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 containing 5% ), keep on ice

after 60min, take 200µl of lipid+cell suspension and add to collection tubes T=60 (containing 200µl of ice-cold containing 5% ), 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