蚊の機能解析のためのカルシウムの生物発光アッセイ（ネッタイシマカ）とダニ（ Rhipicephalus microplus）Gタンパク質共役受容体

The overall goal of the following experiment is to analyze the structure activity relationships of synthesized arthropod neuropeptides on their cognate gpcr. This is achieved by transfection of Chinese hamster ovary, K one empty cells with the desire G PCR R expression vector to obtain stably transformed cell lines derived from a single cell as a second step, the stable cell line is transfected with the quaran plasmid, which carries the reporter Gina Quaran for measuring intracellular bioluminescence in the calcium bioluminescence plate assay. Next, the calcium bioluminescence plate assay is performed in order to determine cytoplasmic calcium levels upon peptide ligand application to these recombinant cells.

Results are obtained that show differences in bioluminescence units upon application of structurally different peptides or different peptide concentrations to the recombinant cells based on elicited intracellular calcium release as detected by the reporter a corrin in the plate assay. The main advantage of utilizing stably transformed clonal cell lines expressing recombinant GPCRs over trends and expression protocols is that receptor expression levels are more uniform, allowing for consistency and results. Further, the targeting of the aquarium protein to the mitochondria allows sensitive calcium measurements through bioluminescence demonstrating the experiment Today will be two graduate students shall in lie, and Simon Kersch as well as a postdoc from my laboratory.

To establish stable cell lines start by growing Chinese hamster ovary, K one empty cells at 37 degrees Celsius in a 5%carbon dioxide humidified incubator. Maintain the empty cells in growth medium with one x antibiotic antimycotic after the cells are growing healthily. See the Chinese hamster ovary K one cells into T 25 tissue culture flasks grow the cells overnight in growth medium without antibiotics until they're about 30%con fluent.

Prior to transfection. Prepare a DNA expression vector containing the GPCR of interest as described in the written protocol. Combine the DNA with 100 microliters of Optum reduced serum medium.

Mix six microliters of lipofection reagent into 100 microliters of F 12 K, serum free medium and incubated room temperature for 30 to 45 minutes. Following incubation, gently mix the DNA and lip expectant solutions in a dropwise fashion. Let the mixture stand at room temperature for 10 to 15 minutes in a 15 milliliter tube.

Gently mix the transfection mixture into 1.8 milliliters of fresh F 12 K medium in a dropwise fashion. Then wash the cells with PBS and add this new transfection solution to the cells. Following an 18 hour incubation.

Change the medium to F 12 K, medium plus 10%FBS without antibiotics and incubate overnight the following day. Split the cells into two T 25 flasks with F 12 K, medium plus 10%FBS without antibiotics as described in the written procedure. Incubate for an additional 18 hours.

Replace the medium with selective medium. Then culture the cells using the selective medium for three to four weeks over time. This will select for cells that have stably incorporated the plasmid into their genomic DNA.

Continue maintaining the cells using maintenance medium periodically freeze the cell lines to prevent loss in case of unexpected contamination. To select for clonal cell lines first trypsin eyes and centrifuge the cells with maintenance medium as described in the written protocol. Then Resus, suspend the cells in five milliliters of maintenance medium.

Remove 0.5 milliliters of the cell suspension and add 4.5 milliliters of fresh maintenance medium to make a 10 x dilution and transfer 100 microliters of the 10 x dilution into 12 wells of a 96 well plate to select for single cells. Continue to make 10 x serial dilution of these cells about 19 times for a theoretical final suspension of one cell per 100 microliters and transfer 100 microliters of each serial dilution into 12 wells of a 96 well plate. After 18 hours of incubation, observe 96 well plates under an inverted light or fluorescence microscope and Mark Wells that appear to contain only one single cell or two cells that have obviously divided from one single cell.

Observe the plates every day changing the medium every three days. Allow the cells to grow for one week until about 80%confluent. Then rinse the marked wells with 200 microliters of PBS and trypsin.

Eyes them with 100 microliters of PBS tripsin EDTA solution. Remove the PBS TRIPSIN EDTA solution. Add 100 microliters of maintenance medium and transfer the cells from each marked well into one well of a six Well plate with one milliliter of maintenance medium.

After growing the cells in the six well plate for three days, transfer the cells into individual T 25 flasks. Periodically freeze the cell line so they can be retrieved if the cells stop performing consistently. To determine the cell line with the highest response, test the transferred cells using the calcium bioluminescence assay with an active ligand such as the receptor, peptide, ligand, or other positive control agonist.

The calcium bioluminescence assay will be demonstrated in detail later in this video. Perform the single cell selection for a second time before proceeding with the experiment. When clonal cell lines are chosen for assays and maintained in culture, it is critical to keep track of passage numbers as high passage number cells may stop performing in a T 25 flask.

Gross cell lines expressing the desired receptor in maintenance medium to about 90%confluence. After tryin and centrifuging the cells resus, suspend them in the maintenance medium dilute cells about 10 x with maintenance medium and count the cell number with a hemo cytometer. Then adjust the cell number to approximately two times 10 to the fifth cells per milliliter seed two milliliters of the cells into each well of a six well plate and incubate the plate for 24 hours after which time the cell should be about 60%confluent prior to the start of the calcium bioluminescence plate assay.

Prepare the reporter gene of interest in the expression vector construct. A mitochondria expressed a quorum report is used here. Then transfect the cells with one microgram of this reporter plasmid as described in the written protocol.

After incubating the cells in a six well plate for 24 hours, trypsin, centrifuge and resuspend, the cells count the cell number to 400, 000 cells per milliliter and transfer 100 microliters into each well of a 96 well white, thin bottom micro titer plate incubate for another 24 hours until about 80%confluence is reached. This is the optimal concentration of cells for the bioluminescence assay. The following steps should be performed in the dark but are shown here in the light.

For demonstrative purposes, prepare enough calcium free DME medium containing five micromolar CHO terezin for 90 microliters per weld. Remove the old media from the plate and add 90 microliters into each. Well incubate the plates for three hours in the dark at 37 degrees Celsius and 5%carbon dioxide after which the cells are ready to be tested.

This demonstration is performed using a Nova star plate reader in bioluminescence mode. To begin purge, the machine pumps the plate should be placed into the plate holder while in the dark. Solubilize the peptides in a 1.5 milliliter EOR tube.

Using enough calcium free DME and media for 10 microliters of 10 x peptide per well. Set the aspirate depth and position determination of the peptide solution before use. Challenge the cells with 10 microliters of the peptide and immediately begin recording the light emission.

In this demonstration, the instrument is set to record the light emission at 465 nanometers for each well every two seconds for a total time of 50 seconds. After the run is complete, wash the machine pumps. Repeat the process for each peptide sample, save the data and wash the machine pumps again following the assay.

The data of light emission from each well is automatically transferred into a Microsoft Excel data sheet by the software program. Paste the data from Excel to Prism Software 4.0. Generate a graph with the various peptide concentrations in the x axis and bioluminescence units.

In the Y axis, select a non-linear regression curve fit analysis to obtain concentration response curves for each peptide in their corresponding EC 50 values. Each experiment should be repeated three times for data analysis. When expressed in Chinese hamster ovary, K one cells, the kinine receptor from the mosquito ad's aegypti behaved as a multi ligand receptor and functionally responded to as low as one animo are.

For the three endogenous eighties kinins tested singly using the calcium bioluminescence plate assay. Statistically different EC 50 values of about 16 nanomolar for eighties aegypti kinine 3 26 nanomolar for eighties aegypti kinine two and 49 nanomolar for eighties aegypti kinin one were determined resulting in a rank order of potency of eighties kinin three as the most potent and eighties kinin one as the least shown. Here is the activity comparison of six alpha amino ISO butyric acid kinin analogs on the tick kinin receptor expressing Chinese hamster ovary K one cell line by a calcium bioluminescence plate assay.

The Y axis represents percent maximal bioluminescence units for each analog expressed as a percentage of bioluminescence observed at a concentration versus the maximal response observed at all concentrations tested for each analog analog KININE HEXA peptide F-F-F-S-W-G amide is the positive control for receptor activity. The single replacement kinine analog double F amino ISO butyric acid WG amide was more active than the positive control HEXA peptide KININE analog, the KININE analog with two aminobutyric acid replacements, aminobutyric acid FF aminobutyric acid WG amide was the most potent of the double replacement analogs tested. After watching this video, you should have a good understanding on how to express A-G-P-C-R and obtain clonal cell lines to analyze structured activity relationships of ligands on receptors by a calcium bio luminescence assay.