The overall goal of this procedure is to investigate how transcellular interactions involving the extracellular domains of specific transmembrane proteins affect beta cell function and more specifically insulin secretion. This is accomplished by first transecting, a mammalian cell line with a plasmid containing the protein of interest in the second step. A beta cell line is co-culture with the mammalian cells.
In the final step, the co-culture is incubated in media with different glucose concentrations in order to enable the assessment of glucose stimulated insulin secretion. Ultimately, insulin radio immunoassays or Eliza's are used to assess the levels of insulin secreted into the media by the cultured beta cells. The main advantage of this technique over existing methods, such as gene overexpression and knockdown, is that it avoids perturbation of the mRNA and protein expression potentially affecting beta cell health and or function in ways that can confound analyses of the specific interaction of the protein.
Begin by transferring the HEC 2 93 cells to a 24 well plate in 0.5 milliliters of HEC 2 93 media per well to ensure that the cells are spread evenly across the bottom of the plate. Shake the plate along one axis at a time with speed synchronous to the wave formed in the well. When the HEC 2 93 cells reach 100%co fluency transfect the cultures with 0.8 micrograms of the plasmid coding for the protein of interest in two microliters of lipo 2000.
According to the lipo protocol, the overall scheme of the co-culture about to be demonstrated is depicted here using a non enzymatic cell remover. Begin this step by harvesting INS one cells at approximately 70 to 80%con fluency from the bottom of a T 75 culture flask. After spinning down the cells for three minutes at 150 Gs at room temperature, use a P 1000 pipette to resuspend the pellet in one milliliter of half INS one and half.
He media then use a 10 milliliter pipette to add an additional 24 milliliters of mixed media and resus suspend the cells aspirate the media from the wells of the 24 well plate containing the HEC 2 9 3 cells. Then using a P 1000 pipette gently add 500 microliters of the INS one cell suspension onto the hex cell layer along the sides of the well. After every six wells, use a 10 milliliter pipette to re suspend the INS one cells.
Again to ensure homogenous cell suspension. Now incubate the cells for 24 to 48 hours, depending on the experimental protocol. After the cells have incubated for the desired experimental period, one well at a time, aspirate the co-culture medium with one hand and immediately replace the media with 250 microliters of 2.5 millimolar glucose in Krebs ringer bicarbonate buffer with the other.
After pre incubating the co cultures for one hour, exchange the low glucose KRB buffer with 250 microliters of either 2.5 millimolar or 20 millimolar glucose. After another hour of incubation, transfer the KRB buffer to micro fuge tubes and spin down the buffer for five minutes at 1500 Gs and four degrees Celsius while the tubes are in the centrifuge. Immediately add 100 microliters of rippa cell lysis buffer with protease inhibitors to the plate and incubate the plate for 20 minutes at four degrees Celsius.
When the tubes have finished spinning, remove 200 microliters of the supernatant for later analysis by insulin radio immunoassay or Eliza. Finally, spin down what is now cell lysate for 15 minutes at 10, 000 gs and then remove 50 microliters of the SNAT for analysis by insulin radio immunoassay or Eliza. Here, the results obtained from co-culture INS one beta cells with HC 2 93 cells transfected to express a neuro liggen isoform referred to here as NLX are shown.
The expression of NLX increased the basal and stimulated insulin secretion by co cultured I NS one cells. The NX extracellular domain must be interacting with another protein or molecule on the I NS one cell surface to cause the INS one cells to secrete more insulin. The results from other types of analyses using the co-culture system are depicted in these next two graphs.
In this first graph, the increase in insulin content of the co cultured INS one cells by NLY is shown. Whereas in this experiment, RNA was harvested from the cell layer and analyzed by R-T-Q-P-C-R. The co-culture with NLY increased the insulin and PDX one mRNA levels.
Following this procedure, additional methods such as QPCR or quantitative immunohistochemistry can be performed to answer additional questions like whether extracellular interactions of the transfected protein can cause changes in cell structure or gene expression.
