HUH7 and A549 control and FADS2 knockdown cells were
grown on glass coverslips (n=4) in low FBS DMEM (1% FBS for HUH7; 0.5% FBS for
A549) for 3 d and subsequently fixed for 15-30 min in 4% PFA at room
temperature. For lipid phase analysis, cells were stained with di-4-ANEPPDHQ
(Thermo Fisher Scientific, MA, USA) according to the manufacturer's
specifications. For imaging, a Nikon A1R confocal microscope attached to Ti
eclipse outfitted with a Plan Apo VC 60x lens oil immersion lens with an NA of
1.4 was used (Nikon Instruments, Tokyo, Japan). The spectral detector was set to
530-590 and 590-650 nm to image the spectral shift of the dye from lipid ordered
to disordered phase. Resulting images were analyzed with NIS software (Nikon
Instruments, NY, USA), thereby segmenting the cells in the images and
calculating the ordered to disordered ratio24 (link). The higher the ordered to disordered ratio, the more saturated
lipids are present in the membrane. Per coverslip, 10 fields of views were
imaged.
grown on glass coverslips (n=4) in low FBS DMEM (1% FBS for HUH7; 0.5% FBS for
A549) for 3 d and subsequently fixed for 15-30 min in 4% PFA at room
temperature. For lipid phase analysis, cells were stained with di-4-ANEPPDHQ
(Thermo Fisher Scientific, MA, USA) according to the manufacturer's
specifications. For imaging, a Nikon A1R confocal microscope attached to Ti
eclipse outfitted with a Plan Apo VC 60x lens oil immersion lens with an NA of
1.4 was used (Nikon Instruments, Tokyo, Japan). The spectral detector was set to
530-590 and 590-650 nm to image the spectral shift of the dye from lipid ordered
to disordered phase. Resulting images were analyzed with NIS software (Nikon
Instruments, NY, USA), thereby segmenting the cells in the images and
calculating the ordered to disordered ratio24 (link). The higher the ordered to disordered ratio, the more saturated
lipids are present in the membrane. Per coverslip, 10 fields of views were
imaged.