Detection of Intracellular Gene Expression in Live Cells of Murine, Human and Porcine Origin Using Fluorescence-labeled Nanoparticles

The overall goal of this procedure is to analyze intracellular gene expression directly in living cells via the application of gene specific fluorescent nanoparticles. This is accomplished by first defining the appropriate target sequences in the gene of interest. The second step is to manufacture nanoparticles with specific capture and reporter strands.

Next, the nanoparticles are added directly to the culture medium of the living cell. The final step is to incubate the cells with the nanoparticles overnight at 37 degrees Celsius and 5%CO2 in a humidified atmosphere. Ultimately, fluorescence microscopy is used to visualize gene specific fluorescence in living target cells while flow cytometry analysis is used to quantify the number of fluorescent cells.

The main advantage of this technique of existing methods such as immunohistochemistry, molecular beacons, or PCR analysis, is that gene expression can be observed visually in live cells without any manipulation of the cell culture. Demonstrating the procedure will be Martina Teresa, a biologist and PhD student from our laboratory, Lynette Henkel, for the flow cytometry. After determining the target sequences, order lyophilized gold nanoparticles from commercially available sources.

These particles are composed of a central gold particle covalently linked to a capture strand, which is bound to a reporter strand. When the target mRNA binds to the capture strand, the reporter strand and its linked fluorophore are released into the solution, allowing the Fluor four to emit fluorescence. Prior to reconstitution.

The nanoparticles should be stored at four degrees Celsius in the dark in a cardboard box. To reconstitute the gold nanoparticles add 20 microliters of double distilled water, resulting in a final concentration of 100 nano molars. The reconstituted nanoparticles can be stored at room temperature in the dark for at least one year.

On the day of application, dilute the stock solution of gold nanoparticles to a 20 x working solution in PBS remove a 24 well plate containing HEK 2 93 cells from the cell culture incubator, aspirate the medium and then add 237.5 microliters of fresh culture medium, then pipette 12.5 microliters of the working solution of nanoparticles into each prepared well and swirl the plate to ensure even distribution of the nanoparticles. Incubate the cells overnight at 37 degrees Celsius and 5%CO2 in a humidified atmosphere. The next day.

Analyze the cell cultures for gene specific site three fluorescence using a standard fluorescent microscope to prepare cells for flow cytometry. Wash the HEK 2 9 3 cells displaying side three induced fluorescence once with PBS and add one milliliter of 0.05%trypsin EDTA incubate the cells for three minutes at 37 degrees Celsius and 5%CO2. After the incubation, add 0.5 milliliters of CO medium and pipette up and down.

Then transfer the cells to a 15 milliliter tube. Add three milliliters of culture medium and centrifuge the tube for five minutes at 300 times G.Next, remove the sate and resus suspend the cells in 200 microliters of ice cold fax buffer. Transfer the cells to a 1.5 milliliter tube and store them on ice in the dark until analysis.

Prior to fax analysis, pass the cells through a 30 micrometer filter to prevent cell aggregation. Then add propidium iodide to the cells at a final concentration of two micrograms per milliliter for at least two minutes, bring the cells to the flow cytometer vortex and load the sample tube onto the device. To detect SI three positive cells.

Use untreated control cells to set the hierarchical gates on the forward scatter or FSC area versus side scatter or SSC area subset, the FSC height versus FSC with subset and the SSE height versus SSE with subset to exclude cell debris and uns scattered cells. Remove the dead cells from the analysis based on propidium iodide staining by displaying the PE channel against the PE Texas Red Channel and then perform the final sort gate on site three PE positive cells. Detection of GA dh.

A constitutively expressed housekeeping gene was performed in HEK 2 93 cells to validate the experimental feasibility. Scrambled negative control nanoparticles show little to no fluorescence while permanently fluorescing positive uptake. Controls show strong signal after overnight incubation.

These nanoparticles were shown to specifically detect the pluripotent stem cell markers, nano and GDF three gene expression in mirroring human and porcine induced pluripotent stem cells or IPCs. Importantly, these markers were only detected in the IPCs and not in the fibroblast cells that served as the feeder layer during IPS cell culture. Both visual and quantitative analysis via flow cytometry were used to assess the labeling efficiency of GA DH nanoparticles graded edition of nanoparticles from zero to 1000.

PICO molar was performed with HEK 2 93 cells, which demonstrated clear concentration dependent increase in fluorescence. Quantitative analysis was then performed in murine embryonic stem cells, which showed approximately 40%CY three positive cells following edition of 400 PICOMOLAR GA DH nanoparticles. Similar values were obtained for the pluripotency markers.

Nano and GDF three Once mastered. This technique can be done within 16 hours if performed properly. After watching this video, you should have a good understanding of how to use gene specific nanoparticles to detect intracellular gene expression directly in living cells using fluorescence microscopy.