Пробоподготовки Стратегии масс-спектрометрии изображений 3D-клеточной культуре Модели

The overall goal of this procedure is to prepare three dimensional cell cultures and analyze by mass spectrometry imaging. This is accomplished by first constructing three dimensional cell cultures and allowing them to grow to the desired size. The second step is to harvest and embed the three dimensional cell cultures in gelatin.

Next, the three dimensional cell cultures are sliced at minus 30 degrees Celsius, using a cryostat and mounted onto conductive slides. The final step is to analyze the slices using a Maldi MSI instrument and perform analysis of the detected molecules. Ultimately, mass spectrometry imaging is used to show the changes in distribution of analytes of many different biomolecule classes across the three dimensional cell culture.

The main advantage of this technique over existing mass like microscopy or auto radiography, is that mass spec imaging allows detection and localization of multiple analyze, such as proteins, lipid drugs, and metabolize in a single experiment without anti labeling. Demonstrating the procedure will be Dorothy Alf Wheat craft, myself, a postdoc and Shin Li, a graduate student from the Hamman lab Prior to starting this procedure, culture and appropriate cell line such as HCT one 16 colon carcinoma cell line in a two dimensional monolayer culture. Following this add 0.19 grams of aros to 10 milliliters of standard media in a 50 milliliter conical tube.

Ensure incorporation by gentle mixing, then autoclave the agros in a water bath for 20 minutes using a multi pipette aspirate. 50 microliters of the Agros media mixture into the 60 central wells of a flat bottomed 96 well culture plate. As the wells at the peripheral edges of the plate have slightly higher evaporation rates at 200 microliters of one x phosphate buffered saline instead of the aros to these edges.

Once the aros mixture has been added to the inner wells, set the plate aside to cool to approximately 37 degrees Celsius before the addition of the cells. At this point, at 200 microliters of the appropriate cell solution to the aros coated 96 well plate cover the plate and set in a humidified incubator with 5%carbon dioxide at 37 degrees cell CS for cell growth. Following incubation, change the media by carefully aspirating the old media from around the small three dimensional cell culture at the bottom of the aros.

Then add 200 microliters of fresh media gently over the three dimensional cell culture. Prepare a solution of approximately 175 milligrams per milliliter of gelatin in high purity water. Mix the gelatin vigorously after the solution has become viscous and difficult to manipulate.

Place the tube in a water bath at approximately 60 degrees Celsius warming until the solution becomes clear and easy to aspirate. Using a two milliliter serological pipette deposit, 0.6 milliliters of the warm gelatin solution into the wells of a 24 well flat bottomed plate. Posit the washed three dimensional cell cultures immediately on top of the gelatin layer using a different two milliliter pipette to avoid rupturing the three dimensional structure.

After the cell cultures are placed on the surface of the gelatin layer, carefully pipette another 0.6 milliliters of the warm gelatin mixture over them so as to not disturb their position. Following this freeze. The gelatin embedded three dimensional cell cultures at minus 80 degrees Celsius.

Once the 24 well plate containing the cell cultures has been removed from the freezer, warm the bottom of the plate with a hand until tweezers can slide gently down the side of the well. Next smoothly. Slide the tweezers around the disc, freeing the gelatin without thawing the center.

Add a drop of water to the cryostat support and adhere the gelatin disc to the support. Then place in the cryostat to freeze. After facing off the excess gelatin to expose the three dimensional cell cultures, slowly slice them with a smooth motion in 12 to 16 micrometer sections.

Once the slices have been thawed, mount them on indium titanium oxide coated glass slides. After labeling the slides, store them in a desiccate for intact protein detection. Wash the slices in cold acetone to remove small molecules such as lipids that could mask the signal.

Prepare the matrix in a solution of organic solvent and water with 0.1%trichloroacetic acid. Use a fine tipped syringe to apply 0.5 microliters of the matrix solution on the three-dimensional cell culture slice. After allowing the matrix to crystallize, dry the slides in a desiccate for at least 30 minutes prior to Maldi MSI.

Analysis at this point, fit the slides in an adapter made to securely hold 75 millimeter by 25 millimeter slides to image the three dimensional cell culture slices. Then load the slide adapter into the instrument. After developing the appropriate data acquisition methods, adjust the mass range for small molecule data acquisition between 101, 000 master charge ratio and for proteins between 8, 020 5, 000 master charge ratio.

Following this, adjust the laser power frequency number of laser shots and spot size using the calibration solution and a nearby sacrificial three dimensional cell culture slice. Save this method for use in the imaging software provided by the instrument manufacturer. Develop protein data acquisition methods for the mass range of choice as previously described.

Then set up the specific MS instrument parameters using the imaging software. After selecting the instrument parameters and setting up the instrument control method position, where to scan over the 3D cell culture. Next, select three appropriate teach points on the sample moving the laser to each.

In turn, obtain the optical photograph from the maldi TOF laser control software using print screen. Then start the imaging process from the imaging software and acquire mass spectra from each slice for targeted analysis. Perform manual analysis of the spectra by clicking a mass in the mean spectrum.

Finally, perform statistical analysis with extracted data sets in mathematical software. Mass spectrometry imaging has the potential to reveal many different molecular distributions in three dimensional cell cultures. Using the previously described method, species from small molecules to large proteins may be tracked across the culture.

After watching this video, you should have a good understanding of how to prepare, slice, and analyze three dimensional cell cultures by mass spectrometry imaging.