TITLE: Coral Lipid Assay for 96-well plates
AUTHORS: Colleen Bove, Justin Baumann

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is designed to work with coral host tissue slurry that has been processed by airbrushing to remove the tissue from the skeleton and after removal of symbiont cells and homoginization. This protocol will allow quantification of lipid concentrations of samples using 96-well plates for rapid assessment. </div><div class = "text-block"><span>This protocol was adapted from Folch et al 1957</span><span style = "vertical-align:super;">1</span><span> and Cheng et al 2011</span><span style = "vertical-align:super;">2</span><span>. For calculation of lipid concentrations, follow the directions presented in the materials section or use the custom Markdown file that can be found on my GitHub (</span><a href="https://github.com/seabove7/protocols/tree/main/LipidAssay_96wellplate" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">LipidAssay_96wellplate</span></a><span>).</span></div><div class = "text-block"><ol style = "list-style-type: decimal;"><li style = "counter-reset:ol0;">Folch J, Lees M, Sloane Stanley GH. A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem. 1957 May;226(1):497-509. PMID: 13428781.</li><li style = "counter-reset:ol0;">Cheng YS, Zheng Y, VanderGheynst JS. Rapid quantitative analysis of lipids using a colorimetric method in a microplate format. Lipids. 2011 Jan;46(1):95-103. doi: 10.1007/s11745-010-3494-0. Epub 2010 Nov 11. PMID: 21069472.</li></ol></div></div>

[STEPS]
?. [Lipid Extraction]
Pull samples from -80 freezer and allow to thaw (maximum of 24 samples per plate in triplicate)
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Vortex sample and transfer 600 μL of coral tissue slurry sample to labelled 1.5 mL tube
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Pre-make new 2:1 (CHCl3:CH3OH) stock every day running plates
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Add 400 μL of CHCl3 and 200 μL of CH3OH in a 2:1 ratio to 1.5 mL sample tube
?. [Lipid Assay Standard Creation]
Make a stock serial dilution in 7 1.5 mL tubes for each plate
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Add 300 μL of CHCl3 to standard tubes 2 through 7
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Vortex then shake on plate shaker for 20 minutes
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Add 160 μL of 0.05M NaCl (CHCl3 : CH3OH : NaCl is in a 2:1:0.8 ratio – keep this ratio)
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Invert tubes gently two times and open and reclose lid
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Centrifuge at 3000 rpm for 5 minutes
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Remove CHCl3 (top) layer and dispose before taking 100 μL for the assay (Do three times for 3 replicates of 100 μL)
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Add 600 μL of 1.5 mg/ml stock to standard tube 1.
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Pull 300 μL from tube 1 and place in tube 2. Back pipette to mix.
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Pull 300 μL from tube 2 and place in tube 3. Back pipette to mix.
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Repeat this process for tubes 3 through 6.
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Discard 300 μL from tube 6 so total volume equals 300 μL.
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Tube 7 is a blank of ONLY CHCl3. DO NOT ADD CORN OILStandard1234567 (blank)(mg/mL)1.50.750.3750.1880.0940.0470.000
Standard1234567 (blank)(mg/mL)1.50.750.3750.1880.0940.0470.000
?. [Lipid Assay]
In a 96-well plate, add 100 μL of sample or standard to each well in triplicate (see template below)123456789101112AStd 1Std 1Std 1S1S1S1S9S9S9S17S17S17BStd 2Std 2Std 2S2S2S2S10S10S10S18S18S18CStd 3Std 3Std 3S3S3S3S11S11S11S19S19S19DStd 4Std 4Std 4S4S4S4S12S12S12S20S20S20EStd 5Std 5Std 5S5S5S5S13S13S13S21S21S21FStd 6Std 6Std 6S6S6S6S14S14S14S22S22S22GStd 7Std 7Std 7S7S7S7S15S15S15S23S23S23HBlankBlankBlankS8S8S8S16S16S16S24S24S24
123456789101112AStd 1Std 1Std 1S1S1S1S9S9S9S17S17S17BStd 2Std 2Std 2S2S2S2S10S10S10S18S18S18CStd 3Std 3Std 3S3S3S3S11S11S11S19S19S19DStd 4Std 4Std 4S4S4S4S12S12S12S20S20S20EStd 5Std 5Std 5S5S5S5S13S13S13S21S21S21FStd 6Std 6Std 6S6S6S6S14S14S14S22S22S22GStd 7Std 7Std 7S7S7S7S15S15S15S23S23S23HBlankBlankBlankS8S8S8S16S16S16S24S24S24
?. [Lipid Assay]
Add 50 μL of CH3OH to each well
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Evaporate solvent on a 90°C hotplate for 10 minutes
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Add 100 μL H2SO4to each well (wells will change from clear to yellow colour)
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Incubate on hotplate at 90°C for 20 minutes
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Cool the plate on ice for 2 minutes
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Transfer 75 μL of each sample or standard from the microplate to a new 96-well microplate (wells may contain sticky residue and bubbles, but you should be able to avoid that to pull the required 75 μL)
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Cover the plate and read background absorbance at 540 nm using microplate reader (this is a baseline measure that is used for correcting the final plate absorbance)
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Add 34.5 μL of 0.2 mg/mL vanillin in 17% phosphoric acid to each well
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Incubate for 10 minutes (should change from yellow to pink color)
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Cover the plate again and read absorbance at 540 nm using microplate reader
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Calculate sample concentrations following method listed under 'Materials'
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All portions of this protocol should be performed in a fume hood as much as possible with appropriate safety precautions, including gloves, lab coat, and closed toe shoes.**The key with this protocol is very careful pipetting**