Chromic acid assay for quantification of total lipids in pollen

Dry down the pollen in a freeze dryer.

Add 1.0 mm zirconia beads to a 2 mL homogenizer tube and fill approximately 1/4 full.

Add 10.0 mg pollen (about 1-2 mg lipid equivalent) to the homogenizer tube.

Add 1000 µL 2:1 chloroform: methanol (Folch solution) to the homogenizer tube.

Pulverize the pollen in the homogenizer tube with the BeadBeater homogenizer for 30 seconds. Tough materials may require additional 30 second rounds of homogenization. Avoid homogenizing for more than 30 seconds at a time to avoid sample overheating. Check the homogenate under a microscope to confirm that the pollen exine wall was fractured.

Turn off the power to the BeadBeater when loading or unloading tubes. Secure the tube caps tightly to avoid sample loss.

Add 210 µL 0.25% KCl to the homogenizer tube and briefly vortex.

Centrifuge the tube contents for 10 minutes at maximum speed in a microcentrifuge.

Two layers will form - an upper aqueous/methanolic layer and a lower chloroform/methanolic layer, with a possible boundary layer at the interface between the two phases. The amount of solvent in these two phases will be different than the two reagents' volumes since some methanol goes into each layer. Estimate the volume of each layer and record it (usually 310 µL upper layer and 690 µL lower layer). Pipette 160 µL of the lower chloroform/methanolic layer into a crimp cap vial being careful not to transfer any solid materials.

Save a second subsample of the lower chloroform layer as backup in case the reaction fails.

Stopping point - The Folch extract can either be dried down immediately for the chromic acid assay or stored capped with a red rubber crimp cap in the -20°C freezer until later analysis.

Turn on the SpeedVac vacuum centrifuge at least 45 minutes before drying samples to allow the solvent vapor trap to cool down before use.

Make a 1.0 mg/mL oleic acid stock solution to create an oleic acid standard curve (0.0 to 1.0 mg/mL step 0.2 mg/mL). Add 9.8 mg oleic acid (11.0 µL) to 9.845 mL 2:1 chloroform: methanol in a scintillation vial and vortex for 10 seconds.

This standard stock solution can be stored sealed in a freezer for up to a few weeks without degradation.

Add oleic acid stock solution and Folch solution to a crimp vial to obtain oleic acid external standards. Make three vial replicates for each concentration.

oleic acid amount of 1.0 mg/mL amount of Folch solution

mg/mL oleic acid added (µL) (2:1 chloroform: methanol) added (µL)

1.0 1000 0

0.8 800 200

0.6 600 400

0.4 400 600

0.2 200 800

0.0 0 1000

Reduce the sample and external standard solutions to dryness in the vacuum centrifuge.

Use dedicated chromic acid equipment, consumables, and safety materials from this point forward.

Take appropriate precautions for chromate handling and waste disposal.

All consumables and equipment that are used during the reaction should be considered contaminated and disposed of as hexavalent chromium waste.

Turn the plate reader on at least 30 minutes before use to allow the reader to heat up.

Take all precautions needed for working with chromic acid from this step forward (see safety warnings). Use specialized protective safety gear and linings to limit contamination. All consumables and benchtop equipment that is used from this point forward should be treated as potentially contaminated by hexavalent chromium and should be stored and disposed of as such.

Make the chromic acid reagent in a sealed glass container with a non-metallic plastic cap (approximately 600 µL per sample or standard (run in triplicate), 200 mL being sufficient for 1000 sample and standards wells on 5 plates). For 200 mL chromic acid, dissolve 1.0 g potassium dichromate in 10.0 mL DI water. Carefully dilute to 200 mL with concentrated sulfuric acid.

Critical - Add the acid slowly to the aqueous dichromate solution while stirring and pause frequently to avoid violent overheating. Do not add water to concentrated acid, as this will likely result in violent boiling over and splashing of this concentrated acid solution. Critical - Add the acid slowly to the aqueous dichromate solution while stirring and pause frequently to avoid violent overheating. Do not add water to concentrated acid, as this will likely result in violent boiling over and splashing of this concentrated acid solution.

Chromic acid can be used for long periods after formation if properly stored in inert containers.

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Add 1.000 mL chromic acid reagent to the crimp vial with the chromic acid-dedicated P1000 pipettor. Be careful not to get any reagent on the vial lip or it will react with the crimp cap. Use the crimper to crimp close each vial with a PFTE aluminum crimp cap. Double crimp the cap at different angles.

Line the hot plate with two layers of aluminum foil to reduce spill damage. Place the crimp-sealed vials in the chromic acid-dedicated heating blocks of the hot plate. Loosely cover the block with aluminum foil to reduce convective heat loss. Heat the vials for 45 minutes at 95°C.

Prepare a working area in a hood that is fully prepared for working with chromate-containing materials and containment of chromate waste. Line all benchtop surfaces that will contain these materials with two layers of benchtop liner paper securely taped down. All containment materials should be disposed of as potentially contaminated after the reaction.

Turn off the heat and wait at least 5 minutes for the heating block to cool. Remove the entire block from the heater and give the block 10 more minutes to cool. Transfer the sample and standard vials to the chromate working area in the hood.

Store the sealed reactant vials in the hood chromate working area until the plate can be made and the absorbance can be read. The chromic acid reaction goes to completion and will not change appreciably over time, but the reaction solution may lose water if the vials are left uncapped for long periods of time. Do not dispose of the vials until you obtain consistent results.

Once cooled, pull the crimped capped reaction vials out of the blocks and place the vials into 24 well plates in the spatial configuration desired for the 96 well plate (3 replicates per sample or standard, 6 standard concentrations and 26 samples per 96 well plate). Standards are usually included on the left side of each plate.

Decap the reaction vials with the decrimper and return each vial to its well plate location. Deposit the decrimped caps into a secondary waste container. Be careful to avoid chromic acid droplets caused by the decapping mechanism.

Load 200 µL of each standard and sample vial reaction solution to a well in a 96 well plate. Make three replicates in triplicate horizontally across the plate for each vial.

Place the plate into the plate reader and read the 620 nm absorbance for each well.

Export the 620 nm absorbance data. Calculate the average 620 nm absorbance for each sample and standard triplicate.

Generate a standard curve from the standards. Calculate the net 620 nm absorbance for each standard concentration as:

ave 620 nm ABS of the standard concentration - ave 620 nm ABS of the 0.0 mg/mL standard

Plot the net 620 nm absorbance for each standard concentration against the standard lipid concentration (mg lipid/mL). Apply a linear best fit line with a zero intercept.

Calculate the net 620 nm absorbance for each sample triplicate as:

ave 620 nm ABS of the sample triplicate - ave 620 nm ABS of the 0.0 mg/mL standard

If sample absorbances indicate that the samples were too concentrated (above 1.0 mg/mL), repeat the plate reading with a 1:4 diluted sample reaction solution (120 µ L sample reaction solution diluted with 480 uL chromic acid reagent, mixed well and run in triplicate). Do not dilute the external standards if diluted samples are rerun.

Construct a standard curve from the average net 620 nm absorbance of each standard concentration. Plot average net 620 nm absorbance of each standard concentration against lipid amounts (in ug) and make a linear best fit line through a zero intercept.

Use the best fit equation to calculate the amount of lipid present in each sample well. Steps that were performed differently between standards and samples (i.e. dilutions, taking only a fraction of the total sample) need corrections in calculations. Make a correction to adjust for the proportion of lower chloroform/methanolic layer taken out of the total volume of this layer (i.e. if 160 µL of 690 µL chloroform/methanolic layer was taken, then 0.23x of the total layer was taken, and the correction would be the inverse (4.31x)). If sample dilutions were performed, correct using the inverse of the dilution factor (i.e. a dilution to 1/5x the initial concentration would be require a 5x correction factor).

Divide the sample total lipid amount by the pollen mass to obtain the total lipid content of the pollen sample.