The overall goal of this procedure is to demonstrate how surface expression of chemos sensory receptors may be detected using live cell staining in HEK 2 93 T cells, this is accomplished by first seating cells on poly D lycine coated cover slips in culture dishes. The second step of the procedure is to transfect DNA in HEK 2 93 T cells. The third step of the procedure is to carry out live cell staining using primary and fluoro four conjugated secondary antibodies.
The final step of the procedure is to fix the stained cells and mount cover slips. Ultimately, results can be obtained that show localization of receptors on the surface of cells through immunofluorescence microscopy. This method enables us to detect chemo sensory receptors exported to the cell surface of heterologous cells like HK 2 93 T.A visual demonstration of this method is going to indicate the key steps that need to be followed closely in order to keep the cells alive for cell self astaining.
Individuals new to this method may find steps like transferring, handling and mounting the cover slip tricky. However, with a few practices, the technique becomes easy With a modified protocol that uses a cgen column, purify plasma DNA of the receptor and the receptor. Transporting protein RT TP one s cloned in a million expression vector PCI ensure that for immunos staining, the receptor coated is end terminally tagged with a 20 amino acid long row epitope to facilitate adherence of the cells.
Code glass cover slips with one milligram per milliliter. Poi de lysine place cover slips in 35 millimeter cell culture dishes coated side up to air dry inside the sterile laminar flow chamber plate HT K 2 9 3 T cells to approximately 30%density in one milliliter of M 10 medium, making sure no air bubble is trapped between the cover slip and the cell culture dish incubated 37 degrees Celsius and 5%carbon dioxide for 18 to 24 hours. For each transfection prepare a DNA mix of 1000 nanograms of receptor construct 250 nanograms of RTP one s construct, and 50 nanograms of GFP construct.
As per manufacturer's instructions, transfect the cells with lipectomy 2000. Prepare staining media by adding sodium azide and hippies to M 10. This may be stored at four degrees Celsius for several months.
Next, prepare washing buffer of 15 millimolar sodium azide and 10 millimolar heis. In cold HBSS, the washing buffer may be stored at four degrees Celsius for several months. In order to carry out the staining in the cold line a tray with parfum and place on ice for five minutes.
With the cell side up transfer the glass cover slips containing transfected cells to the paraform from the corner of the cover slip Carefully add 100 microliters of cold primary antibody diluted in staining media. Incubate for 60 minutes at room temperature. Now wash the cells in the original 35 millimeter culture dishes by alternatingly adding and aspirating cold washing solution.
Repeat twice. Similarly, incubate the SI three conjugated secondary antibody for 30 minutes. Perform washes three times in the 35 millimeter dishes.
Finally, fix the cells with chilled 1%Paraform aldehyde solution. Taking care to exclude all air bubbles. Mount the cover slips cell side down on microscope slides using mole rinse the surface of cover slips with distilled water to remove salt deposits.
Typically, as with the OL FFR 62 and RTPs one cell surface staining of receptors is characterized by punctate appearance of fluorescent signal from the conjugated antibody Once mastered, this technique can be used to stain 32 40 cover slips in as little as two and a half to three hours While attempting this procedure with multiple cover slips, it's important to transfer and start antibody incubation one cover slip at a time to prevent cells from drying out.
