preparation of op50 for caenorhabditis elegans culture

Hochdurchsatz-Assay zur Messung der Nahrungsaufnahme und Lebensdauer von Caenorhabditis elegans

Caenorhabditis elegans has been extensively used in aging research. &#160;It has been a powerful model to study the genetics underlying metabolism-mediated longevity induced by dietary restriction, reduced mitochondrial activity, or reduced insulin signaling.1,2,3,4,5,6,7. However, measuring feeding in the context of longevity has been proven difficult for C. elegans due to the small size of the animal3,8,9,10,11,12,13,14,15.
Feeding is a fundamental biological behavior required for survival, and its tight regulation lies at the core of maintaining metabolic health, reproduction, and aging. Feeding behavior is controlled by multiple signaling pathways in both the nervous system and periphery and, thus, can only be studied in vivo16,17,18,19. Feeding represents the first of three pillars: (i) energy intake, (ii) energy storage, and (iii) energy expenditure that govern an organism&#39;s energy homeostasis. Feeding in C. elegans has been chiefly investigated by measuring the pharyngeal pumping, as defined by the rate of contractions of the pharynx. This approach has provided crucial insights into C. elegans&#39; feeding behavior3,4,8,12,13,20,21; however, pharyngeal pumping, especially measured over short intervals of minutes, is not necessarily correlated with food intake.
Food intake is not only determined by the pharyngeal pumping rate but also by parameters such as duration and frequency of feeding bouts and the density of food bacteria. An animal may have a very high pharyngeal pumping, but the length of its feeding bouts may be shorter, offsetting the increased rate. Another confounding factor is the efficiency by which pumping may or may not ingest and grind up bacteria. An extreme example of uncoupling food intake from pharyngeal pumping is the addition of serotonin without any food (bacteria) present. In the presence of exogenous serotonin, animals pump at a high rate, but without bacteria present, the high pumping rate does not lead to food intake2,6,13,22,23.
The bacterial clearance method presented here measures the food intake of worm populations in individual wells as the decline of bacterial concentrations over time (Figure&#160;1). This approach is similar to those used in other species where the disappearance of food represents a measure of food intake10,11,24,25,26,27,28. In the original publication, we validated this method of measuring food intake by feeding C. elegans with 15N isotope-labeled bacteria11. Subsequent measures by mass spectrometry showed that measuring the disappearance of bacteria correlated strongly with the occurrence of isotope labeling, revealing the actual uptake of the bacteria into the animal11. Thus, we are confident that the bacterial clearance assay represents food intake in most circumstances. The assay&#39;s purpose is not to replace the pharyngeal pumping assay but to add to the toolbox of assays to study feeding and metabolism in C. elegans and how it relates to aging and longevity.

Autor im Rampenlicht: Erforschung des Zusammenhangs zwischen Lipotoxizität und HFpEF 

Quantifizierung der Nahrungsaufnahme bei Caenorhabditis elegans durch Messung der bakteriellen Clearance

My research focuses on understanding the transcriptional and metabolic pathways underlying drug-induced changes in feeding behavior. I am working to identify drugs that lower food intake and extend lifespan indicative of activating healthy fasting pathways. Using our bacterial clearance assay, we identified two distinct pathways that modulate serotonin-induced food intake and antipsychotic-induced food intake.I am now studying how these pathways relate to feeding in long-lived mutants. This protocol allows researchers to study the relationship between feeding and longevity. The feeding rate is usually characterized by pharyngeal pumping.However, the pumping rate in C.elegans is not necessarily proportional to the amount of food being eaten. When added to those previous assays, this protocol gives a quantifiable amount of food intake per worm population. Our assay allows us to quantitatively modulate feeding using small molecules, and to determine how the induced change alter lifespan simultaneously.

Aufbereitung von OP50 für Caenorhabditis elegans Kultur

Zu Beginn inokulieren Sie eine einzelne OP50-Kolonie in fünf Millilitern LB mit Ampicillin und Amphotericin B und inkubieren Sie etwa sechs Stunden lang bei 37 Grad Celsius in einem Bakterienschüttler. Sobald die Kultur trüb wird, verdünnen Sie die Präinokulumkultur von OP50 um 1 bis 2.000 in 250 Millilitern TB mit Ampicillin und Glycerin. Anschließend wird die OP50-Flüssigkultur in ein steriles Zentrifugationsröhrchen überführt.Zentrifugieren Sie das Röhrchen 15 Minuten lang bei 3.100 g in einer Tischzentrifuge bei vier Grad Celsius. Entsorgen Sie den Überstand und resuspendieren Sie das OP50-Pellet in sterilem Wasser, bevor Sie es rezentrifugieren. Nach der zweiten Wäsche den Überstand entsorgen und das OP50-Pellet in sterilem Wasser auf ein Volumen von 50 Millilitern resuspendieren.Füllen Sie es in ein vorgewogenes konisches 50-Milliliter-Röhrchen um. Zentrifugieren Sie das Röhrchen 20 Minuten lang, um den OP50 zu pelletieren. Entfernen Sie am Ende der Zentrifugation vorsichtig alle verbleibenden Überstände und stellen Sie sicher, dass kein Wasser im Röhrchen verbleibt.Berechnen Sie das Gewicht des Pellets, indem Sie das Gewicht des leeren Zentrifugationsrohrs vom Gewicht des Zentrifugationsrohrs mit dem Pellet abziehen. Resuspendieren Sie das OP50-Pellet gründlich in S-vollständigem Puffer, um eine Konzentration von 100 Milligramm pro Milliliter zu erreichen. Testen Sie die OP50-Suspension auf einer Agarplatte, um sie auf Verunreinigungen zu überprüfen.Die OP50-Suspension ist in einem S-vollständigen Puffer bei vier Grad Celsius zu lagern.

preparation-of-op50-for-caenorhabditis-elegans-culture

Research

JoVE Journal

Behavior

Preparation of OP50 for Caenorhabditis elegans Culture

532 Aufrufe. Zu Beginn inokulieren Sie eine einzelne OP50-Kolonie in fünf Millilitern LB mit Ampicillin und Amphotericin B und inkubieren Sie etwa sechs Stunden lang bei 37 Grad Celsius in einem Bakterienschüttler. Sobald die Kultur trüb wird, verdünnen Sie die Präinokulumkultur von OP50 um 1 bis 2.000 in 250 Millilitern TB mit Ampicillin und Glycerin. Anschließend wird die OP50-Flüssigkultur in ein steriles Zentrifugationsröhrchen überführt.Zentrifugieren Sie das Röhrchen 15 Minuten lan...

Serie: Aufbereitung von OP50 für Caenorhabditis elegans Kultur

Quantifying Food Intake in Caenorhabditis elegans by Measuring Bacterial Clearance

Feeding is an essential biological process for an organism&#39;s growth, reproduction, and survival. This assay aims to measure the food intake of Caenorhabditis elegans&#160;(C. elegans), an important parameter when studying the genetics of aging or metabolism. In most species, feeding is determined by measuring the difference between the amount of food provided and the amount left after a given time interval. The method presented here uses the same strategy to determine the feeding of C. elegans. It measures the amount of bacteria, the food source of C. elegans, cleared within 72 h. This method uses 96-well microtiter plates and has allowed the screening of hundreds of drugs for their ability to modulate food intake at a speed and depth not possible in other animal models. The strength of this assay is that it allows to measure feeding and lifespan simultaneously and directly measures the disappearance of food and, thus, is based on the same principles used for other organisms, facilitating species-to-species comparison.

This protocol describes an assay to quantify Caenorhabditis elegans feeding rate based on measuring the clearance of bacteria in liquid culture.

Feeding is an essential biological process for an organism&#39;s growth, reproduction, and survival. This assay aims to measure the food intake of ...

Forschung

Verhalten

To begin, inoculate a single OP50 colony in five milliliters of LB with ampicillin and amphotericin B and incubate for approximately six hours at 37 degrees Celsius in a bacterial shaker. Once the culture becomes cloudy, dilute the preinoculum culture of OP50 by 1 to 2, 000 in 250 milliliters of TB with ampicillin and glycerol. Then transfer the OP50 liquid culture into a sterile centrifugation tube.Centrifuge the tube for 15 minutes at 3, 100 G in a tabletop centrifuge at four degrees Celsius. Discard the supernatant and resuspend the OP50 pellet in sterile water before recentrifugation. After the second wash, discard the supernatant and resuspend the OP50 pellet in sterile water to a volume of 50 milliliters.Transfer it to a pre-weighed 50 milliliter conical tube. Centrifuge the tube for 20 minutes to pellet the OP50. At the end of the centrifugation, carefully remove all remaining supernatant, ensuring no water remains in the tube.Calculate the weight of the pellet by subtracting the weight of the empty centrifugation tube from the weight of the centrifugation tube with the pellet. Thoroughly resuspend the OP50 pellet in S-complete buffer to achieve a concentration of 100 milligrams per milliliter. Test the OP50 suspension on an agar plate to check for any contamination.Store the OP50 suspension in S-complete buffer at four degrees Celsius.

Synchronous Caenorhabditis elegans Culture Preparation and Measurement of Bacterial Clearance for Food Intake Assay

To begin, select a nematode growth medium or NGM plate occupied by starved L1 stage Caenorhabditis elegans larvae. Using one milliliter of sterile water, wash the worms off the plate gently. Aliquot approximately 300 microliters of the worm population onto NGM plates seeded with concentrated OP50.Allow the plates to dry using a plate dryer or a Bunsen burner, and then incubate at 20 degree Celsius until a significant population becomes gravid adults. To collect the worms, wash them off the plate with up to 10 milliliters of sterile water and transfer a warm water mixture into a 15-milliliter conical tube. Allow the worms to settle by gravity at the bottom of the tube for approximately four minutes.Using a small pipette tip, carefully aspirate and discard the supernatant. Then add up to 15 milliliters of sterile water. After the final wash, remove the supernatant and add five milliliters of a freshly prepared solution of bleach and sodium hydroxide.Incubate the worms for five minutes at room temperature while vortexing every minute for at least 10 seconds. Monitor the progress using a dissecting microscope. Once all the adults break open or dissolve, add M9 buffer to neutralize the reaction, bringing the final volume to 15 milliliters.Then centrifuge the tube for two minutes at 1, 300 G.Wash the eggs two more times with 13 milliliters of M9 buffer via centrifugation followed by a single wash with 15 milliliters of S complete buffer. Then centrifuge the eggs for two minutes at 1, 300 G.After centrifugation, aspirate the supernatant and add 10 milliliters of S complete buffer. Rotate the tube gently at room temperature overnight using a mutator or a similar device.Using a dissecting microscope, assess if the worms have hatched. Count the worms in 10-microliter drops of S buffer under the microscope. Prepare a liquid worm mixture with 60 worms per milliliter in S complete medium containing carbenicillin, amphotericin B, and OP50.Add 120 microliters of medium with worms to rows A to G and no worms medium to row H.Then seal the plate with a tape sealer to prevent contamination and evaporation. Incubate the sealed plates for approximately 65 hours at 20 degrees Celsius until the animals become L4 worms. Add 30 microliters of a 0.6-millimolar fluorodeoxyuridine stock solution to each well to sterilize the animals at the L4 stage.Reseal the plate using tape sealers and agitate it for 20 minutes at 800 RPM on a microtiter plate shaker. Return the plates to the 20-degree-Celsius incubator. The following day, add the drug of interest to the day one culture.Reseal the plates with tape sealer and shake for 20 minutes at 800 RPM on a plate shaker. Then after removing the lid and seal, measure the OD600 in a plate reader for the day one measurement. Reseal and return the plates to a 20-degree-Celsius incubator.Using an inverted microscope, preferably with a two times objective, count the worm population in each well and record the numbers in a spreadsheet. After counting, return the plates to the 20-degree-Celsius incubator. The dose response curve's a full change in food intake as a function of serotonin concentration showed that the N2 strain can overeat in a dose-dependent manner.The daf-16 mu86 mutant exhibits higher basal feeding than N2.However, it cannot respond to serotonin in the same dose-dependent manner as the N2 strain. A significant difference was observed in the food intake of worms treated with loxapine but fed with bacteria killed by either x-rays, gamma rays, or paraformaldehyde. Comparison of the food intake of a series of genetic strains showed that exc-4 and cgr-1 mutants eat less while srp-6 mutants eat more.