Fluorescent Measurement Of Lipid Contentin The Modelorganism Chlamydomonas Reinhardtii And Mutants Screening

ThegreenalgaChlamydomonasreinhardtiiisoneofthemoststudiedmicroalgae,whichhasthe potentialtobeusedasamodelsystemtostudylipidmetabolism.Establishmentofamethodinthi sorganismforrapidandsimplemeasurementofneutrallipidsisdesirable.Fluorescentmeasure mentofneutrallipidsbyNileRedstaininghasbeenwidelyusedinvariouscelltypesincludingmi croalgae.However,asystematicstudyofNileRedstainingtomeasureneutrallipidsinChlamyd omonashasnotbeenreported.Here,weshowthatNileRedstainingissuitableforrelativeandabs olutequantificationofneutrallipidsaswellasforpossiblelarge-scalescreeningformutantsdefe ctiveinlipidaccumulation.The cellular neutral lipids were detected and quantified though a96-wells plate with ELIASA. We scanned the excitation spectrum and emission spectrum and found the optimized excitation and emission wavelength as528nm and576nm, specially. Still, we comparedandoptimizedthefactorsinvolvedNileRedstainingincludingsolvents,cellconcent ration, stainingtime,and working concentration of NileRed.Wedeterminedthat5%DMSOwithlμgmL-1NileRedand5-15mintimewindowafterstainingwasoptimalformeasuringlipidcontentofcellswithintheran geoflto8×106cellsmL-1, and the protection from light are not necessary. Spectrum methods though Nile red staining were used to quantify the content of neutral lipid. We developed the standard method and standard addition method with the staining of Nile red and estimated the lipid content with flourescence intensity. The triolein was used as the standard to quantify cellular neutral lipid in spectrum methods and quantification with thin layer chromatography. The methods used in staining were followed by the protocol we had bulit before. There was no distinct difference between the spectrum methodsand traditional gravimetric determination method. Inaddition,wedevelopedaprotocolthatcouldbepotentiallyusedforlargescalescreeningforce llswithdifferentlipidcontent.Isolation of mutant cells with different lipid content is key to study the machanism of lipid metabolic or nitrogen pathways. Two different simulation experiments of mutants with disordering of formation of lipid drops were designed to validate screening method feasibility. And we have found some mutants successfully including three ones with deficiency of lipid drops, one with overmuch lipid drops and some other type mutants, compared with the normal widetype cells. ThuS,theworkreportedhereprovidestimelyneededtechniquestofacilitateChlamydomonast obeusedasamodelorganismforstudyinglipidmetabolismforbiodieselproduction.