The overall aim of this procedure is to identify signaling pathways that regulate changes in functional beta cell mass. This is accomplished in isolated rat eyelets by over expressing or suppressing the expression of a gene using adenoviral vectors. After this manipulation, beta cell function is assessed by insulin secretion and eyelet replication measured by tritiated thymidine incorporation.
Ultimately, these assays can show whether a gene of interest regulates beta cell proliferation and or function in vitro. This method can help answer key questions in the eyelid biology field, such as does a particular component of a signaling pathway influence beta cell growth and function? Prepare a six well non tissue culture coated plate by adding two milliliters of modified media to the required number of wells.
For example, a typical experiment may require three wells, one each for a no virus control, a virus control, and the experimental group warm the plate to 37 degrees Celsius in a tissue culture incubator for at least 30 minutes. Immediately following rat eyelet isolation, place one to 200 eyelets in each wells of the prepared plate. 60 eyelets will be used for biochemical assays and the remaining eyelets can be used for gene expression Studies or immuno blotting gently swell the plate to bring the eyelets to the center of each.
Well then immediately pipette the adenovirus directly onto the eyelets. In the center of the dish. Use one to 500 multiplicities of infection depending on the effectiveness of the adenovirus at over expressing or knocking down your protein of interest.
Efficient adenoviral penetration to the core of the eyelid increases the consistency of results. Transducing the eyelids immediately following eyelid. Isolation is essential.
Do not disturb the plate for five minutes, then move it to the incubator for 24 hours. The next day, remove the plate from the incubator and gently swell the eyelets to the centers of the wells. Transfer the eyelets to a new well containing fresh media using a 200 microliter micro pipette.
If the eyelets become attached to the plate, they can be gently dislodged with the pipette tip. It is helpful to use a control virus expressing GFP to verify adequate transduction efficiency by using confocal microscopy to visualize penetration of the adenovirus into the islet core culture. The islets for one to three more days, changing the media daily.
The time of culture must be optimized by pilot studies and varies based on the experimental goals. For the final 24 hours, incorporate tritiated thymidine into the media at a concentration of one micro curie per millimeter of media. First freshly prepare 50 milliliters of the working SAB in a 50 milliliter conical tube and warm it up in a 37 degrees Celsius water bath.
Pipette 10 milliliters of working SAB into a 15 milliliter conical tube and add 66.8 microliters of 2.5 molar D glucose. To prepare the high glucose SAB, add 44.8 microliters of 2.5 monolo D glucose to the remaining 40 milliliters of the working SAB. To prepare the low glucose SAB next label three 1.7 milliliter micro centrifuge tubes.
For each well of the six well plate and add one milliliter of PBS to each tube. Do not forget to follow institutional protocols for the handling and disposal of the radioactive eyelets. Using a stereoscope or standard microscope, select and transfer 20 comparably sized eyelets into each micro centrifuge tube.
For example, each tube may contain five small, 10 medium, and five large sized eyelets. Although the results are normalized to total protein content at the end of the experiment, it is critical to sizeism match the eyelets between groups. After allowing the eyelets to settle on the bottom of the tube by gravity or by centrifugation, aspirate, weigh the PBS with a micro pipette and then proceed with preparing the eyelets for the biochemical assays.
To prepare the eyelets for the insulin secretion assay, they must first be pre incubated with low glucose SAB. To do this, add 400 microliters of the low glucose SAB to the tubes and place them with caps on into the tissue culture incubator For 60 minutes after an hour, aspirate away the pre incubation low glucose SAB To measure basal insulin secretion. Repeat the pre incubation procedure.
However, to measure stimulated insulin secretion, use 400 microliters of high glucose SAB instead of low glucose SAB and as before, incubate the eyelets for 60 minutes after incubation in either high or low glucose SAB aspirate and the SAB for the insulin radio immunoassay, which will be executed according to the manufacturer's protocol. Then proceed with the thymidine incorporation assay using the same collection of eyelets. Begin with the eyelets just used for the insulin secretion assay.
Add one milliliter of PBS and allow the eyelets to settle by gravity. Then aspirate the PBS with a micro pipette. Discard and repeat this step once more.
Next, add 500 microliters of ice cold trichloroacetic acid to the eyelets and incubate them on ice for 30 minutes. Centrifuge the tubes at four degrees Celsius. Then aspiration, discard the TCA.
Next, add 80 microliters of 0.3 normal sodium hydroxide and incubate the eyelets for 30 minutes of room temperature. During this incubation vigorously vortex the samples for five to 10 seconds every 10 minutes. In parallel, add four milliliters of a conno safe counting cocktail to seven milliliter liquid scintillation counting tubes.
When the incubation of the eyelets is over, add 50 microliters of the eyelets to a scintillation tube. Shake the capped tube briefly and measure the samples radioactivity in a liquid scintillation counter. Also to measure the protein concentration, transfer 10 microliters of vilas to a commercial bionic acid assay and follow the manufacturer's protocol.
Later during data analysis, normalize the results to the protein concentration using the described protocols. Eyelet replication and beta cell function. In rat eyelets were assessed an adenoviral overexpression of hypothetical gene six robustly stimulated eyelet replication without altering beta cell function.
Increased expression of gene six, increased DNA synthesis as measured by the incorporation of thymidine because most of the cells in the rat eyelet are beta cells. Further experimentation could show that this increase in thymidine incorporation is due to an increase in beta cell replication. The insulin secretion assay demonstrated the overexpression of gene six did not alter one of the primary beta cell functions insulin secretion at low and high glucose.
The increase in insulin secretion at low and high glucose concentrations reflects the health of the eyelets after treatment with adenovirus. If increasing the expression of gene six impaired beta cell function, this would likely be reflected as a decrease in the insulin secreted at high stimulate re glucose concentrations. After watching this video, you should have a good understanding of how to measure beta cell replication and insulin secretion to determine if a particular gene influences beta cell growth or function.
