The object of this experiment is to determine existence of junction between cells by using scarp loading.
Gap junctions are channels having essential functions, cell–cell transfer of ions and small molecules among cells. They are a kind of commutation system between cells to regulate cell processes. (1, 2)
Gap junctions are formed by a kind of family of protein known as connexion. These proteins have 21 different isoforms and together generate connexons or hemichannels, protein complexes, forming junction channels with neighbour cells. A cell can produce various isoforms according to their specialization and it can cause formation of channels. All connexons include 4 transmembrane proteins with N-terminus facing cytosol, a C-terminus, two extracellular loop domains. By aligning with adjacent cells, these connexons from a cell forms gap junctions among cells. These junctions serve passage of small molecules and ions to be regulated and kept processed cellular functions with adjacent cells for organism. (1, 2 and 3)
Scientists use some methods to detect these gap junctions such as electrophysiological measurements, microinjection, fluorescent dye uptake, and scrape loading. Scientists can know existence of gap junction between cells and level of permeability of gap junction. (4)
This approach is based on a simple method. On cell culture, a cut is formed and dye is loaded onto cells. If cells have junction among each other, dye are transmitted by using this junctions. This method can show simply existence or present of linkage between cells. Concentration of transmitted dye also can show level of permeability of cells. Scrape loading can serve an observation about present of junctional linkage of cells quantitatively.(5)
Material and Method
The medium was kept from plate including cells and put into a tube to save for next steps. Cell culture was washed with 1000µl 0f 1xPBS for 2 times and then with 500µl of 1xPBS for 1 time by micropipette. 1µl of Neurobiotin was added onto cells as primary antibody. 4 cuts were formed on the plate and then the sample was left to stand for 10 minutes for diffusion of it at RT (room temperature) while plate was covered. Cells were washed with 1xPBS for 3 times. Next, the medium we removed at first step was added and was waited for 15 min at RT. While washing with PBS for 3 times, at each washing step cells were waited with 5 min intervals for removing excess materials in the plate. Cells have been fixed with 500µl PFA and they were waited for 15 min at RT. By washing with PBS for 3 times, PFA was removed from the media. Permeability of cells was increased by adding 500 µl TrintonX and it was waited for 10 min for effectiveness of TrintonX. PBS was used for removing TrintonX from media for 3 times by washing cells. Streptavidin and DAPI were transmitted into plate as 500 µl-solutions. The cells were allowed to stand for 30 min so that the dye could be held. Finally, cells were washed 3 times with PBS at 15 min-intervals.
In this experiment, we purposed to observe gap junction between MCF10A cells. MCF10A cells are normal epithelia cell line. These cells are suitable for this experiment because they have frequent and close level. Also they can give a high cell concentration for a good observation. If all conditions of experiment are good, we can expect differences of levels of concentration of dye. Scrape loading method works on simple way. A cut can be line for dye can expend from cell to cell. By using this approach, we can observe degree of gap junctional link quantitatively. We used secondary antibodies (Streptavidin), be seeing as red color, for investigate passage dye and DAPI (blue) for showing nucleus of cells because of attachment of DAPI to nucleotides. DAPI was used for determining existence of space between cells or sufficient cell concentration. However, when observations are made, it can be seen is no passage of molecules between cells (Figure 1 and 3). If Figure 2 and 4 are examined, it can be seen that there is no problem in distance between cells for substance passage. The problem for passage can be dyes. The dyes couldn’t have been attached and it can cause that we can’t have a good results. The other reason can be because of the approach for these cells. Scrape loading method is not inappropriate to observe for small cells or cell assemblies as well as low-density. If junctional or degree of link is so small, or dyed cells are screened as induvial, this method can be complicated. (5, 6)
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