The aim of this procedure is to provide a rapid and reproducible approach to investigate how antibodies, auto molecular mechanisms related to neuronal function and the pathogenesis of neurologic disease. First antibodies are labeled with an ATO five 50 NHS fluorescent label. Next, the antibodies are mixed with the transfection reagent then transfected into neuronal cells.
In culture, 48 hours following transfection neurons are fixed mounted with DPI media and visualized by immunofluorescence microscopy. Transfection efficiency can be calculated using imaging software. This method can help answer key questions in neuroscience and neuroimmunology, such as how autoantibodies alter molecular mechanisms related to neuronal function and the pathogenesis of immune mediated Neurologic disease.
We first had the idea for this method when we wanted to test the hypothesis that antibodies to h and RPA one and RNA binding protein over expressed in neurons contributed to mechanism of neurodegeneration demonstrating the procedure will be Josh Douglas, a Graduate student Before beginning the procedure. Ensure that high quality intact neuronal cells plated at 100, 000 cells per well, and at least 80%cofluent are available the day before the experiments. The day of the assay observe cells to ensure that they are healthy.
Begin by preparing a 0.1 mil of sodium bicarbonate buffer by mixing 8.4 grams of sodium bicarbonate and 29.2 grams of sodium chloride in one liter of water. Then adjust the buffer's pH to 8.4 to 500 microliters of buffer. Add 70 microliters of the antibody to be transfected in this case, anti H-N-R-N-P-A one and 35 microliters of the labeling reagent here at oh five 50 NHS.
Rotate the solution at room temperature for one hour, then inject it into a dialyzer and dialyzed in two liters of PBS at four degrees Celsius overnight. The next day use a spin column to concentrate the dialyze labeled antibody. Finally, determine the antibody concentration using a NanoDrop spectrophotometer.
In an eight well slide see 10, 000 cells per well in a volume of 500 microliters of complete DMEM. Incubate the cells at 37 degrees Celsius overnight or until the cell co fluency reaches at least 70%once the cells are 70%cofluent. Begin the procedure by mixing two microliters of antibody, delivering the transfection reagents and two micrograms of labeled antibody at 0.5 micrograms per microliter in an einor and incubate the solution for 10 to 15 minutes at room temperature.
In the meantime, remove the SUP natum from the cell culture wells and replace it with exactly 394 microliters per well, a fresh medium. Once the transfection solution is ready, add 100 microliters of DMEM and mix by pipetting up and down. Then add exactly 106 microliters of transfection mixture to each well containing cells of obtaining a final volume of 500 microliters per well.
Be sure to include an UNT TRANSFECTED negative control well in which DMEM rather than antibody is added. Incubate the cells for 48 hours at 37 degrees Celsius. Following this incubation, replace the medium with fresh DMEM, perform live imaging using a fluorescent microscope 48 hours after labeling.
To visualize ATO five 50 NHS labeled antibodies, use the SI three filter after live imaging aspirate off the medium and fix the cells by adding 4%paraldehyde and incubating the slide for 15 minutes at room temperature. Once the cells are fixed, wash them four times in PBS allowing a five minute incubation period in PBS for each wash. Then remove the PBS and add a drop of mounting medium containing DPI.
To each, well gently place a cover slip on top of the cells using a fluorescent microscope. Begin imaging the cells. In this experiment, we performed live fluorescent cell imaging of neurons successfully transfected with ATO five 50 NHS labeled anti H-N-R-N-P-A one antibodies.
This image shows antibodies within the neuronal cells, including the cytoplasm neuronal processes, as well as the nucleus fixation and nucleus staining. Using DPI shown here in blue allows the transfection efficiency to be determined by dividing the number of transfected cells by the number of total cells. In this case, the efficiency is 50%Following this procedure.
Other methods like immuno cyto chemistry, RNA, extraction, microray analysis, and western blots can be performed in order to answer additional questions such as colocalization of antibodies within cellular organelles, as well as changes in neuronal function and integrity related to the specificity of the antibody for the target antigen. In this video, we have shown how to label antibodies with a fluorescent label, prepare a complex of transfection reagent with fluorescently labeled antibodies, transfect neurons, and calculate transfection efficiency. This method is useful in the evaluation of how autoantibodies affect cellular functions such as viability, apoptosis, neuronal transport, as well as the molecular mechanisms related to neuronal function and the pathogenesis of neurologic disease.
