The overall goal of the following experiment is to assess the contributions of colonization and its associated damage to the initiation of innate immune responses in sea elegance. This is achieved by exposing a synchronized population of young adult sea elegance to pseudomonas arosa expressing green fluorescent protein or GFP, which produces a population of worms variably colonized by the pathogen. After washing the differentially, colonized populations of worms are separated into pure populations focusing on the two extremes of fully colonized and non colonized worms.
Next RNA is extracted from these populations and immune gene expression is evaluated in comparison to worms grown on non-pathogenic e coli. In order to examine the relationship between the immune response and colonization, results are obtained to show that the innate immune response of sea elegance to p arosa can be decoupled from pathogen colonization. The main advantage of this technique over other approaches for studying initiation of immune response in sea elegance is that it addresses the variability in infection in warm populations, allowing us to tease apart different aspects of the infection process.
This method can help answer key questions in sea elegance, innate immunity such as what is the basis for initiation of innate immune responses and in a high throughput manner. What's the impact of different genetic or environmental manipulations on the ability to resist pathogen colonization? To begin grow worms on several nematode, growth medium or NGM plates seeded with e coli bacteria until many worms have reached the grave stage.
Then treat grave animals with egg prep solution to obtain a synchronized culture. Next plate. The eggs on several 60 millimeter NGM plates seeded with bacteria at a density of roughly 150 to 200 eggs per plate.
A minimum of 15 to 20 plates will be needed to obtain several thousands of worms. For the sorting experiment, users should scale up by using larger diameter plates with more eggs per plate if larger numbers of worms are needed. Incubate the plates at 20 degrees Celsius for two days until worms have reached the L four or young adult stage.
On the next day of the egg prep inoculate A GFP expressing POSA culture, PA 14 GFP, into two milliliters of kings B.Medium containing antibiotic. Incubate the culture at 37 degrees Celsius with agitation overnight. The next day plate the PA 14 GFP culture onto as many slow killing plates as needed for the assay on each 150 millimeter Petri dish.
Pipette 75 to 100 microliters of saturated culture and spread evenly. Using a sterile glass spreader, incubate the plates at 37 degrees Celsius for 20 to 24 hours. Remove the plates with PA 14 GFP from the incubator and allow them to cool to room temperature.
Meanwhile, to collect the worms grown on NGM plates, add a small amount of M nine buffer enough to cover the plate. Gently swirl the plate and transfer the liquid to a 15 milliliter conical tube. Allow the worms to settle to the bottom and remove excess liquid before transferring the worms onto PA 14 GFP plates In a minimal volume of liquid, when using 150 millimeter plates, several hundred worms can be placed on a single plate.
Incubate the plates at 25 degrees Celsius for 18 to 21 hours for p arosa exposed young adult worms. This is the time window displaying the optimal distribution of colonized versus non colonized animals before performing the sample sort ensure that the machine is functioning properly. Essential steps are outlined here and details can be obtained online.
Set the sheath valve pressure to approximately five PSI to verify that the sheath fluid flow rate is appropriate. Place a 15 milliliter conical tube beneath the dispenser. Turn on the sheath valve and turn off the sorter valve.
Hold the tube beneath the dispenser for 60 seconds. The volume in the tube should be nine to 10 milliliters. If it is outside this range.
Adjust the sheath valve pressure accordingly to ensure that the sheath fluid flow rate will allow accurate sorting. Test the sort rate by first turning off the sheath valve. Then add about 25 milliliters of control particle solution to the reservoir.
Turn on the sheath valve and the sample valve on the computer software. Navigate to the control particle mode and click acquire. Make sure the particle flow rate is approximately five to eight particles per second with the time of flight approximating the width of the 40 micrometer particle.
This will ensure that only a single 40 micron object is passing through at a time. If values are outside of this range, adjust to settings accordingly. Using M nine buffer wash worms into 15 milliliter conical tubes, allow adult worms to sink to the bottom of the tube by gravity and remove the supernatant.
Fill the tube with fresh M nine buffer. Repeat this process three to four times. This removes a large proportion of larvae as well as excess bacteria and takes about 10 minutes.
Add worms to the worm sorter reservoir containing a gently mixing small stir bar. Adjust the initial signal gain by setting the green photomultiplier tube to 600 and the red and yellow photomultiplier tube to 200. Begin acquiring data to adjust the settings.
Aim for a sort rate of 25 to 30 events or worms per second. If the number rate is too high, dilute the worms in the reservoir with M nine. Buffer accordingly.
If the rate is too low, add more worms in a small volume of M nine. If the experiment requires a very stringent separation of colonized versus non colonized worms, set the coincidence check to pure. This ensures that if two objects occur too close to each other or in the same drop, the machine rejects the drop.
Rather than collecting it to sort the population of interest, draw gates around the axis indicating size and fluorescence intensity. The values for fluorescent gating must be determined empirically. Next place a multi-well plate or Petri dish underneath the dispenser to collect the worms.
Then open the sort valve to begin sorting worms. Collect a small population of animals to verify under the microscope that the sorted population is the population of interest. If not, adjust a gating parameters accordingly and continue with sample collection.
When age matched genetically identical sea elegance that are exposed to GFP expressing posa result in a wide distribution in levels of colonization efficient separation of non colonized from colonized worms was achieved using a worm sorter. Unlike non colonized worms, colonized worms show overt signs of such as sluggishness and reduce defecation. The latter may be a reason why worms colonized by P arosa have difficulty clearing infection.
Four genes were induced similarly to P arosa exposure regardless of the colonization status of the worms and quantitative R-T-P-C-R was used to measure expression of the genes. The four genes are known to be part of the CL egan's immune response. Lys two encodes a lysozyme two uncharacterized genes response specifically to infection of which one also contributes to infection resistance, and PGP five responds to infection as well as to heavy metal stress.
The results corroborate previously published results indicating that colonization and its associated damage are not required for an immune response. Instead, pointing at pathogen associated molecular patterns as the signal data showing that PGP five is induced in non colonized animals, which are not expected to be experiencing. Extensive damage suggests an overlap in the regulatory programs, controlling immune responses and general stress responses in sea elegance.
This method can provide new insights into initiation of immune responses in sea elegance, but it could also be applied to other questions such as the effects of environmental conditions or genetic manipulation on colonization or competition between different pathogens besides infection processes. This method could also be applied to follow other interactions of worms with their environment such as ingestion or sequestration of metabolites or chemicals. Following this procedure, the sorted populations of interest can be used for a variety of purposes beyond gene expression.
These include collecting live animals for subsequent infection or survival assays or behavioral experiments. This technique provides a useful means to study the effects of pathogen exposure and colonization on innate immunity and sea elegance.I.
