Published: March 30th, 2013
We present robust biochemical and microscopic methods for studying Caenorhabditis elegans lipid stores. A rapid, simple, fixing-staining procedure for fluorescent lipid droplet imaging leverages the spectral properties of the lipophilic dye Nile red. We then present biochemical measurement of triglycerides and phospholipids using solid phase extraction and gas chromatography-mass spectrometry.
The nematode C. elegans has emerged as an important model for the study of conserved genetic pathways regulating fat metabolism as it relates to human obesity and its associated pathologies. Several previous methodologies developed for the visualization of C. elegans triglyceride-rich fat stores have proven to be erroneous, highlighting cellular compartments other than lipid droplets. Other methods require specialized equipment, are time-consuming, or yield inconsistent results. We introduce a rapid, reproducible, fixative-based Nile red staining method for the accurate and rapid detection of neutral lipid droplets in C. elegans. A short fixation step in 40% isopropanol makes animals completely permeable to Nile red, which is then used to stain animals. Spectral properties of this lipophilic dye allow it to strongly and selectively fluoresce in the yellow-green spectrum only when in a lipid-rich environment, but not in more polar environments. Thus, lipid droplets can be visualized on a fluorescent microscope equipped with simple GFP imaging capability after only a brief Nile red staining step in isopropanol. The speed, affordability, and reproducibility of this protocol make it ideally suited for high throughput screens. We also demonstrate a paired method for the biochemical determination of triglycerides and phospholipids using gas chromatography mass-spectrometry. This more rigorous protocol should be used as confirmation of results obtained from the Nile red microscopic lipid determination. We anticipate that these techniques will become new standards in the field of C. elegans metabolic research.
Conservation of metabolic pathways between humans and the nematode Caenorhabditis elegans makes it a powerful model organism for the study of obesity. While C. elegans do not have adipocytes dedicated to fat storage like in mammals, they do store triglycerides in lipid droplets1 and possess many of the same regulators of fat storage and energy use2,3. To assay for fat levels in C. elegans, several methods have been proposed with varying levels of ease and success. The once common use of fluorescent, vital dyes4-7 was recently called into question, and these reagents proved to be staining a compartment distinct from the main fat storage depot of C. elegans1,8-11 . Fixative-based non-fluorescent dyes, such as Sudan black and Oil-red-O, while successful at highlighting lipid droplets in the worm12-14, do not have as large a dynamic range for quantitation as fluorescent dyes8,13,15. Furthermore, current methods of fixation for both fluorescent and non-fluorescent dyes are time-consuming and involve multiple freeze-thaw steps and/or the use of toxic chemicals1,9,10 . The use of specific BODIPY-labeled lipid analogs has been reported to highlight lipid droplets when fed to living worms with short-term labeling15. However this relies on uptake of the dye and is therefore biased by C. elegans handling and metabolism of BODIPY fatty acid analogs. An established alternative to lipid-staining dyes is label-free, coherent anti-Stokes Raman scattering (CARS) or stimulated Raman scattering (SRS) microscopy, which take advantage of the characteristic vibrational properties of lipid molecules to visualize fat stores10,16,17 . However, expensive specialized equipment is necessary for this method, and throughput is low at best.
To fulfill a need for rapid, scalable analysis of neutral lipid stores in C. elegans metabolic research, we introduce a novel, highly reproducible method of fixative-based Nile red lipid staining. Spectral and physicochemical properties of the lipophilic dye Nile red induce a yellow-gold-spectral shift in its excitation-emission peak, allowing it to fluoresce in the green emission spectrum only when in a lipid-rich environment, but not in more polar environments18,19 . Thus lipid droplets can be detected after simple staining by the use of a green fluorescent protein (GFP) filter set for fluorescence microscopy. The ease and affordability of this technique make it ideally suited for high throughput screens. We also demonstrate a paired, rigorous method for biochemical determination of triglycerides and phospholipids using solid phase extraction and gas chromatography-mass spectrometry. Biochemical lipid measurement correlates with Nile red-based microscopic determination of C. elegans fat mass, and should be used as a confirmation of findings made with microscopic lipid determination.
1. Preparation of Rectangular Amp/Tet (A/T) LB Agar Plates and NGM IPTG RNAi Plates
A/T plates should be prepared 1-4 weeks prior to use and stored in the dark at 4 °C.
Prepare 96-well RNAi plates 3 days up to 2 weeks in advance.
2. Stamp Frozen Library Plates onto Rectangular Amp/Tet (A/T) LB Agar Plates
3. Grow RNAi Bacterial Clones Overnight in Deep-well Blocks
4. Seed RNAi Bacterial Clones onto 96-well NGM RNAi Plates
5. Prepare a Synchronous Population of Worms
Two 10 cm plates filled with many gravid worms are used for egg preparation for every 6 96-well RNAi plates. Be sure worms are non-starved for at least two generations (7 days).
6. Add Worms to RNAi Plates
7. Fix and Stain Worms in Nile Red
8. Wash and Image Worms on a High-throughput Microscope
9. Biochemical Lipid Determination Using Solid Phase Extraction (SPE) and Gas Chromatography-Mass Spectrometry (GC/MS)
For validation of lipid levels based on Nile red fluorescence, gas chromatography followed by mass spectrometry analysis (GC/MS) can be performed on fatty acid methyl esters (FAME) derived from triacylglycerols (TAG) and phospholipids (PL) from worm pellets. Steps involving organics should be performed under a fume hood. Care should be taken that lipids are kept under nitrogen protected from light during extended storage or incubation steps as they are very vulnerable to oxidation. Worm pellets from 2,500-5,000 synchronous animals are used as input, prepared exactly as above except that animals are grown on 10 cm RNAi plates.
|Hold 2 min
|Ramp 10 °C per min
|Hold 4 min
|Ramp 5 °C per min
|Hold 3 min
|Ramp 10 °C per min
|Hold 5 min
A 3 min solvent delay is used at the MSD to minimize wear on the filament. Thereafter, masses between 50 and 550 Daltons are detected with the MS quadruple set at 150 °C and the ms source set at 230 °C, with a thermal auxiliary temperature of 270 °C.
An example of an imaged plate that has been taken through the fat staining workflow is shown in Figure 1. Specific controls for high fat (daf-2 insulin receptor RNAi), low fat (lpd-3 protein required for lipid storage granules in the gut RNAi), small and low fat (fasn-1 fatty acid synthase RNAi), and empty vector (control) show the variations in fluorescence intensity according to the fat levels of the animals, and the corresponding lipid amounts determined by GC/MS analysis (Figure 2). Note that gene inactivations leading to developmentally arrested, small animals will often appear to have much lower fat than adult control animals, irrespective of any connection to fat metabolism (Figure 3). Secondary analyses, including staining analysis and lipid biochemistry with SPE-GCMS of wild-type control animals of a similar developmental stage must be done to ensure the validity of small or developmentally arrested positive hits. In the case of fasn-1 RNAi (Figure 2), animals arrest in either the L2 or L3 larval stage, and have fat staining lower than adult control RNAi, but similar in abundance to L2-L3 stage control RNAi animals (fat stain percent of adult control RNAi: 49.9 ± 4.5% for fasn-1 RNAi and 20.2 ± 2.1% for L2 control RNAi, and 64.7 ± 1.3 for L3 control RNAi). Alternatively, biochemical analysis indicated a lower TAG/PL ratio than L2 stage matched N2 control animals (TAG/PL: 22.5 ± 2.9 for fasn-1 RNAi and 46.1 ± 3.4 for stage-matched control RNAi, P ≤ 0.05). Thus in this instance it can be confirmed by biochemical analysis that fasn-1 has a role in lipid storage rather than just a stage-specific reduction in fat mass.
The follow-up analysis of imaged worms relies heavily on a computational platform such as Cell Profiler using the WormToolbox30. For smaller numbers of images, a publicly available image analysis platform such as ImageJ, freely available from the NIH, may be used. For our analysis, unique Matlab scripts were used first to identify the well, and subsequently to exclude empty wells or those with bubbles, and distinguish small debris from worms. Manual quality control was necessary for images flagged for exclusion for the reasons mentioned above. We found empirically that 90th percentile intensity of worm pixels across a well, normalized to worm area across a well, provided consistent metric of staining intensity at all stages of worm development (Figure 3). Integrating total fat mass over the area of the worm, on the contrary, would tend to score small bright worms equivalently with large dim worms. Because our method does not integrate total fluorescent signal over the area of the worm, in taking an intensity percentile, changes in worm size per se do not bias the intensity measured. Instead, differences in the pixel intensity distribution are measured. However, in spite of this, pronounced differences are seen in staining pixel intensity distribution between animals of different developmental stages, so it is important to study the effect of RNAi to any gene against animals of a comparable developmental stage (Figure 3). Average variability between replicates is shown in Figure 4 indicating high reproducibility of the fat staining method. Note that the strength of the method is highly dependent on obtaining quality images for each plate, under uniform illumination conditions, using identical exposure times, and with minimal bubbles, debris, or crossover of worms into other wells.
SPE was performed on pre-mixed, purified standards to determine the degree to which TAGs could be separated from PLs (Figure 5A). In this analysis, we achieved a 100% separation of TAG and PL, indicated as a complete absence of 17:0 fatty acids derived from PL in the TAG sample and a corresponding complete absence of 13:0 fatty acids derived from TAG in the PL sample (Figure 5A). Thus SPE is highly efficient at separating lipid classes. This analysis does not indicate that all TAGs of different chain lengths will be purified equivalently by SPE and detected by GCMS with similar efficiency. It only assures that TAG and PL lipids in total are completely separated from each other.
A sample GC-MS trace of N2 wild type animals taken through solid phase extraction and FAME preparation is shown in Figure 5B. For both TAG and PL, peaks can be identified based upon retention time and parent and daughter ions on the mass spectrum, and the total area under the identified peaks normalized to the area of the respective internal standards. To determine the normalized amount of TAG, the area under all peaks of the TAG chromatogram, except the 13:0 standard, were added together and divided by the area under the 13:0 peak. Likewise, to determine the normalized total PL amount, the area under all peaks, except the 17:0 standard, were added together and divided by the area under the 17:0 peak. The normalized values for the TAG and PL samples are then used to calculate the final TAG/PL ratio for the sample:
It should be noted that the data from SPE-GCMS permit a much more sophisticated analysis of fatty acids present in each lipid fraction than simple calculation of the total TAG to PL ratio. For example, the investigator can integrate a single peak corresponding to a single fatty acid and compare its normalized abundance across samples (e.g. comparing in N2 wild-type versus daf-2 RNAi the integrated area of the 18:0 peak divided by the 13:0 standard peak, normalized to total PL mass divided by PL 17:0 standard peak area):
Should the investigator choose, it would also be possible to determine the percentage of any given fatty acid in the TAG or PL fractions as a percentage of the total amount of fatty acid quantitated (as an example 18:0 in the TAG fraction in N2 worms):
Figure 1. Typical workflow for the overall experiment. Day 1, frozen RNAi library plates are stamped onto Amp/Tet LB agar plates and incubated at 37 °C. Day 2, Amp/Tet plates are used to seed liquid cultures of RNAi clones in deep well blocks. Day 3, bacteria are concentrated by centrifugation and transferred to 96-well RNAi plates. Day 4, L1 hatchling C. elegans are added to RNAi plates in liquid and allowed to dry. Day 7, animals are collected in liquid, stained with Nile red in 40% isopropanol, mounted on 96-well Teflon slides, and imaged with a high-throughput microscope.
Figure 2. Fixative-based Nile red stains major C. elegans fat stores. Representative bright field and GFP fluorescence images are shown of N2 worms fed on fasn-1 (small, low fat), lpd-3 (low fat), control (empty vector), or daf-2 (high fat) RNAi clones. Samples were processed as per the protocol (schematic in Figure 1). Animals fixed, stained in Nile red, and imaged result in comparable lipid levels as obtained from biochemical lipid measurement. Isopropanol-fixed Nile red fluorescence levels, acquired from at least 6 biological replicates, are shown in the first row. Quantitative triglyceride levels, reflective of overall neutral lipid stores when normalized to overall phospholipid levels (TAG/PL), taken from 4 biological replicates, are shown in the second row. It is important to note that the absolute quantitation achieved from fixative Nile red staining and biochemical lipid determination often do not match perfectly. In this instance, though qualitatively similar, fasn-1(RNAi) has more different triglyceride mass vs. control by biochemical methods than indicated by microscopy, lpd-3 is comparably different, and daf-2(RNAi) is less different, though all differences are highly statistically significant and measurements were highly reproducible. Data shown are mean ± SEM (*, P ≤0.01; **, P ≤ 0.0001 relative to control, determined by unpaired, two-tailed T-test assuming equal variance).
Figure 3. Fat mass determined by fixation Nile red staining at different larval stages. Animals were grown on OP50 bacteria and collected at the L1, L2, L3, L4 and gravid adult developmental stages. Images were captured following fixation staining and analyzed using a customized Matlab script to determine the 90th percentile of pixel intensity over the identified worm area. Note that the lipid levels of animals greatly increase as the animal develops from L2 to L4 stage, and then decrease slightly as the animal begins to divert calories toward the proliferation of the germline in the adult stage. Data shown are mean ± SEM for 6-8 replicates (**, P ≤ 0.0001 relative to gravid adult, determined by unpaired, two-tailed T-test).
Figure 4. High reproducibility across independent biological replicates. Shown is a scatter plot of Nile red fluorescence intensities across biological replicates (n = 6-8). For each replicate, all values are normalized to the mean intensity of the empty vector controls. Data shown are mean ± SEM (**, P ≤ 0.0001 relative to control, determined by unpaired, two-tailed T-test assuming equal variance).
Figure 5. GC-MS chromatograms of SPE separated standards and wild type TAG and PL abundance. (A) GC-MS traces are shown of the SPE separation of TAG and PL standards. Note the complete absence of 13:0 fatty acid in the PL trace and the corresponding absence of the 17:0 fatty acid in the TAG trace, indicating complete separation of TAG (tritridecanoin) from PL (1,2-diheptadecanoyl-sn-glycero-3-phospho-(1'-rac-glycerol)). (B) A representative full spectrum of TAG and PL from a sample of N2 wild type animals fed vector control RNAi (HT115 bacteria with L4440 empty vector RNAi). One microliter of sample was injected into an Agilent 6890/5973N GCMS according to the protocol, and individual peaks were identified by retention time and their respective mass spectra. Abbreviations: cyclo, cyclopropane fatty acid; iso: iso-methyl branched chain fatty acid; GLA, gamma linolenic acid; ALA, alpha linolenic acid; DGLA, dihomo gamma linolenic acid; EPA, eicosapentaenoic acid. Click here to view larger figure.
The presented protocol allows for rapid, large scale screening of lipid levels in C. elegans, which can be easily adapted to high throughput screens. Unlike previously used methods, which involve a lengthy fixation step in paraformaldehyde followed by several freeze-thaw cycles8,10 , our protocol requires a simple 3-min fixation in isopropanol. We have found that by staining C. elegans in 40% isopropanol, they become freely permeable to Nile red, without the use of additional freeze-thaw or fixation steps. Also, as Nile red selectively fluoresces in hydrophobic compartments in the green spectrum18,19,31 , no destaining is necessary. In total, animals can be taken from RNAi plates to stained and ready to image animals in just over 2.5 hr. This method can be performed relatively inexpensively and with high reproducibility. The method does not depend upon the ability of C. elegans to uptake or concentrate the dye, and therefore does not have the same limitations as feeding based methods using vital dyes. Given reproducibility and precision of measurements, the method is suitable for screening large numbers of gene inactivations by RNAi. If quantitation of lipid levels is desired based upon fixative Nile red staining, we recommend the use of 4-6 biological replicates with the appropriate controls and statistical testing applied.
It is likely that many gene inactivations leading to small animals (due to RNAi clones causing delayed or arrested development) would come out of a screen for fat regulators, regardless of any connection to fat metabolism. While the image analysis program we utilized accounts for worms of different sizes by normalizing the fluorescence intensity to the worm area, individual researchers may also want to consider comparing the fat staining levels of small animals to wild type animals of a similar developmental stage. In the case of small or developmentally arrested animals with apparent low fat mass, we suggest that no single modality is sufficient to demonstrate altered lipid metabolism. Several follow-up experiments, including SPE-GCMS, could be required to ascertain the lipid-regulatory function of these genes. For known metabolic regulator fasn-1, SPE-GCMS analysis compared to developmentally matched N2 L2 control animals was necessary to confirm the apparent low fat phenotype found when compared to adult control animals. While Nile red fat staining indicated lower fat levels relative to control adult RNAi, when compared to stage-matched L2 control RNAi animals, no apparent difference was seen. Accordingly, use of a second method, i.e. biochemical quantification of the TAG/PL ratio was necessary to determine that fasn-1 is a true regulator of fat mass, as fasn-1 RNAi is biochemically distinguishable from stage-matched L2 control RNAi animals.
While we present imaging and processing for a high-throughput platform, a conventional upright microscope can easily be used to capture images from worms mounted on agarose pads20 or on conventional sized Teflon-printed 8 to 12 well microscope slides. The only limitation is that as the green fluorescence of lipid droplets stained with Nile red photobleaches quickly, systematic, uniform exposure conditions need to be used to ensure comparability between images. We find that a fully automated microscope with uniform image acquisition conditions works best to this effect.
One of the great strengths of this protocol is that after images are collected they may be saved for future re-analysis. The same images could be used to determine body size in addition to fat content. Moreover, as computational programs become more advanced, there is the prospect for single worm analysis, generating statistically significant results from a single well. Thus our method that utilizes high-throughput microscopy has some advantages over published screen methodologies that use a Copas Biosort to quantify fluorescent signals32,33 . However, the methodologies are definitely complimentary.
Precise, biochemical determination of lipid content by SPE and GC/MS confirms the Nile red staining of the major fat stores in C. elegans. Although the absolute quantitation achieved from fixative Nile red staining and biochemical lipid determination often do not match perfectly, both methods produce highly reproducible, statistically significant results that agree qualitatively. In addition, this method can be tailored to meet the specific needs of individual researchers who may require further analysis of the separate classes of glycosphingolipids, phospholipids, or triglycerides present in various samples. It should be noted that other groups have used technology other than SPE to separate lipid classes prior to GC/MS22. Thin layer chromatography (TLC) and high pressure liquid chromatography (HPLC) has advantages over SPE in that they might be able to better separate distinct neutral lipids (e.g. TAG, diacylglycerols, free fatty acids, and cholesterol esters) from polar lipids (phosphatidylcholine, phosphatidylethanolamine) and glycol-sphingolipids. We choose SPE because it requires a minimum of starting material (2,500-5,000 worms), it emphasizes the major long-term energy stores, TAGs, which make up most of the neutral lipid fraction4, and is rapid, requiring no specialized equipment or plates. It should be emphasized that based upon standard mixtures, SPE completely separates TAGs from PLs, and is highly reproducible and reliable (see Figure 5A). While quantitation and analysis of fatty acid methyl esters requires a GC/MS, this equipment is commonly available, and if not locally, samples may be generated as above and stored at -80 °C under nitrogen until analysis by a collaborator or commercial GC/MS service is arranged.
The GCMS output contains high-resolution data on each fatty acid within each lipid species. This permits the determination of detailed differences between samples compared. As an example, the investigator could determine whether the levels of certain fatty acids were altered in abundance more significantly than others in daf-2, lpd-3 or fasn-1 RNAi versus vector control. This enormously powerful tool can therefore provide detailed information on which lipid species are altered in abundance with any experimental manipulation.
For our analysis, we most often normalize TAG mass to PL mass. While protein or DNA could also be used to normalize lipid mass following determination from an aliquot of sonicated worm pellet, we find that these methods are subject to introduced human error during all pipetting steps and in sample recovery from each extraction. It is for this reason that we chose to instead use PL mass as our normalization factor. Unnatural standards introduced at the start of the protocol (tritridecanoin for TAG, 1,2-diheptadecanoyl-sn-glycero-3-phospho-(1'-rac-glycerol) for PL) are carried through all steps of the solid phase extraction and FAME preparation, and guarantee quantitative and representative recovery of both TAG and PL fractions. The same guarantee cannot be made if investigators use protein or DNA to normalize TAG mass. We believe PL to be the most robust gauge by which to compare TAG levels across samples, as it has been previously shown that PL is only minutely altered by the same stimulus effecting large mass shifts in TAG levels, e.g. extended starvation or increased exercise34-36. PL are thought to play a structural role, ensuring membrane fluidity and cellular integrity and signaling roles rather than as energy storage37-39. In our hands, the TAG/PL ratio most closely matches what is obtained by fixative-based lipid staining with Nile red, and agrees with that found by other groups21. An alternative strategy is used by the Watts laboratory, where the percentage of lipid TAG is calculated versus total lipids9,22,29 .
The use of non-native types of TAG and PL standard ensures that no native fatty acids will be included in the normalization calculation. The choice of chain length is purely based upon the need of these standards to not interfere with the mass spectra of native fatty acids. We have found that fatty acids of 13:0 and 17:0 serve best to this purpose. It is possible, given the different chemical properties of shorter chain fatty acids, that our unnatural fatty acid standards might not be representative of recovery of all chain-length fatty acids or chains with different saturation indices. Thus it is possible that during SPE or GCMS we might see differences in the efficiency of purification or detection between lipids of different chain lengths. Based upon this and the different ionization of different fatty acids in the GCMS, it is not possible to make an absolute statement about the abundance of any given fatty acid. Therefore, we only compare between samples within a single experiment abundances of any given fatty acid. If one wanted to absolutely quantitate a fatty acid, one manner this could be achieved would be to run a sample prepared as above spiked with a known amount of a stably isotopically labeled 13C version of that or similar fatty acid, so that it could be distinguished based upon mass of the parent ion in the MSD, for example. Given that we are only comparing relative amounts of lipid classes or any given fatty acid, our 13:0 TAG and 17:0 PL standards serve this purpose adequately, at a minimum of cost, to assure adequate and representative recovery of each lipid class.
Until this point, there has been a lack of consensus as to the interpretation of certain results obtained by various methodologies, and to what the prevailing methodologies should be. Overall it should be noted that no one method in isolation is ideally suited to study fat metabolism in C. elegans. However, by using multiple screening and validation modalities, one can achieve confidence in data obtained on lipid regulatory genes. Thus, we intend for our protocol to serve as a benchmark for future work in the field of C. elegans fat research.
The authors have no conflicting interests to disclose.
We would like to thank Michael Kacergis for technical assistance and Lianfeng Wu for discussions and reading the manuscript. This work was supported by a career development award from the NIH/NIDDK (K08DK087941 to A.A.S.), a NIH/NIDDK training grant (5T32DK007028 to E.C.P.), the Ellison Medical Foundation New Scholar in Aging Award (to A.A.S), and the Charles H. Hood Foundation Child Health Research Award (to A.A.S.).
|Name of the reagent
|Biohit M Line
|M10, M100, M300
|Biohit E Line
|1 M KH2PO4
|pH 6.0 Sterilized by Autoclaving
|1 M MgSO4
|Sterilized by Autoclaving
|1 M CaCl2
|Sterilized by Autoclaving
|5 mg/ml Cholesterol
|In 100% Ethanol
|200 mg/ml Carbenicillin
|Filter Sterilized 0.2 μ
|1 M IPTG
|Filter Sterilized 0.2 μ
|100 mg/ml Ampicillin
|Filter Sterilized 0.2 μ
|5 mg/ml Tetracycline
|In 100% Ethanol
|96-well TC Plates
|For 96-well RNAi
|Rectangular A/T Plates
|For stamping RNAi
|Prepared as per manufacturer instructions
|96-well Deep Well Assay Blocks
|Rectangular V-bottom assay block
|96-well Shallow Well Assay Block
|Round V-bottom assay block
|Sterile Sealing Film
|Aluminum Sealing Film
|Thermowell Sealing tape for 96-well plates
|See Hope et al.20
|See recipe below
|96-well Teflon Masked Microscope Slides
|Can also order Trevigen 96-well Cometslide
|96-well Gold-Seal Coverslides
|Can also use second 96-well Cometslide
|Deep-well PCR plate
|Non-sterile adhesive film
|DM6000 fully automated with Prior 96-well stage
|With Chroma 49002 GFP filter set and 2x objective
|Avanti Polar Lipids
|Wiretrol 50 μl Pipet
|SPE Silica Column
|Hypersep SI, 100 mg resin bed
|Solid Phase Chromatography Manifold
|Supelco Visiprep 24-DL
|10 ml size
|Borosilicate Pasteur Pipets
|Borosilicate Culture Tubes
|Threaded Borosilicate Tubes
|8 ml capacity
|Phenolic Caps for Culture Tubes
|GC/MS Autosampler Vials
|Caps for Autosampler Vials
|6890 GC / 5973 MSD
|Outfitted with a Supelcowax-10 column
Nematode Growth Media (NGM): for 25 96-well RNAi plates, prepare 1 L of NGM:
Rectangular Ampicillin/Tetracycline LB Agar Plates (A/T): for 20 A/T plates, prepare 1 L of media:
LB bacterial growth media: for 4 L, sufficient for growing and resuspending 25 96-well blocks
Mix 62 g LB powder (US Biologicals), 4 L distilled water, and 4.8 ml 2 M NaOH. Autoclave in 1 L or 0.5 L aliquots, liquid cycle, 40 min. Can be stored up to 1 year in airtight bottles at room temperature. Discard if cloudy.
100 mg/ml Ampicillin
Mix 5 g ampicillin powder and deionized water to a total volume of 50 ml. Filter sterilize 0.2 μ, dispense in 1 ml aliquots and freeze at -20 °C until use.
5 mg/ml Tetracycline
Add 1.25 g tetracycline powder to 250 ml 100% ethanol. Mix well until fully dissolved and store at -20 °C in a light-tight container.
200 mg/ml Carbenicillin
Mix 10 g carbenicillin powder and deionized water to a total volume of 50 ml. Filter sterilize 0.2 μ, dispense in 1 ml aliquots and freeze at -20 °C until use.
5 mg/ml Cholesterol
Add 1.25 g cholesterol to 250 ml 100% ethanol. Mix well and store at 4 °C for up to 6 months.
1 M MgSO4
Dissolve 120.37 g MgSO4 in 800 ml deionized water. Bring final volume to 1 L. Autoclave 40 min liquid cycle and store at room temperature.
1 M CaCl2
Dissolve 110.98 g CaCl2 in 800 ml deionized water. Bring final volume to 1 L. Autoclave 40 min liquid cycle and store at room temperature.
1 M KH2PO4, pH 6.0
Dissolve 136.09 g KH2PO4 in 800 ml deionized water. pH to 6.0 and bring final volume to 1 L. Autoclave 40 min liquid cycle and store at room temperature.
M9 Buffer: for 1 L—we routinely make 8-10 L batches
Dissolve 3 g KH2PO4, 6 g Na2HPO4, 5 g NaCl, 1 ml 1 M MgSO4 in 900 ml. Bring final volume to 1 L. Aliquot in 100-500 ml bottles and autoclave 40 min on liquid cycle and store at room temperature.
10 N NaOH
Dissolve 400 g NaOH in 800 ml deionized water, allow to cool and bring to a final volume of 1 L. Store in 50 ml tubes at room temperature.
1 M IPTG
Add 23.81 g IPTG to deionized water and bring total volume to 100 ml. Filter sterilize 0.2 μ, dispense in 1 ml aliquots and freeze at -20 °C until use.
Egg Preparation Bleach/NaOH Solution: 20 ml sufficient for one egg preparation
Add 4 ml bleach, 1 ml 10 N NaOH, 5 ml H2O, and 10 ml M9 buffer to a 50 ml polypropylene conical tube. Make immediately before use.
Nile red stock solution
Add 50 ml 100% Acetone to 25 mg Nile red powder. Mix well and store at room temperature in light-tight parafilmed container. Can be stored for months in this manner.
Nile red staining solution: for 500 ml, sufficient for staining 30 x 96-well plates
Add 3 ml Nile red stock solution to 500 ml 40% v/v isopropanol. Store at room temperature in foil-wrapped glass bottle. Prepare immediately before use.
Weigh 7.6 mg phospholipid standard, 1,2-diheptadecanoyl-sn-glycero-3-phospho-(1'-rac-glycerol), in a glass beaker. Dissolve in 20 ml 2:1 chloroform:methanol (25 nanomoles per 50 μl). Keep covered with aluminum foil until use. Standard can also be kept at -80 °C in an airtight glass vial with phenolic cap for 1 year.
Weigh 6.8 mg triglyceride standard, tritridecanoin, into a glass beaker. Dissolve in 30 ml 2:1 chloroform:methanol (16.7 nanomoles per 50 μl). Keep covered with aluminum foil until use. Standard can also be kept at -80 °C in an airtight glass vial with phenolic cap for 1 year.
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