The overall goal of this procedure is to perform multiple viability assays that provide a more comprehensive view of cellular fitness than any one assay can afford by itself. This is accomplished by first measuring the A TP levels of plated cells in order to assess metabolic function. The second step is to immunostain cytoskeletal proteins such as alpha tubulin or micro tubular associated protein two, using infrared antibodies with wavelengths of 800 nanometers.
Next cells are labeled with infrared drac five and sapphire to stain the nucleus and cytoplasm at 700 nanometers. The final step is to image the alpha tubulin or map two and the drac five plus sapphire at both the 700 and 800 nanometer wavelengths on an infrared imager. Ultimately, these luminescent and infrared assays provide information on both anatomical structures and physiological function.
The main advantage of these techniques over existing methods such as the MTT viability assay, is the rapid and sensitive measurement of multiple aspects of cellular integrity. These methods can help answer key questions in the fields of neuroscience and pharmacology, such as whether therapeutic compounds can protect anatomical structures and metabolic function. To begin harvest and reflate cells in 100 or 200 microliters of media at varying cell densities in a 96 well plate as described in the accompanying protocol, fill the surrounding wells with 200 microliters of sterile water to reduce edge evaporation effects.
Let the cells adhere overnight at 37 degrees Celsius in a CO2 buffered incubator. The next day, remove 50 out of the 100 microliters of media or 150 out of the 200 microliters of media so as to leave 50 microliters of media behind in each well. Next, add 25 microliters of the cell titer glow reagent to each of the interior wells in columns two through six.
To obtain a one to two dilution in columns seven through 11 rows B through D, add 50 microliters of the cell titer glow reagent to obtain a one to one dilution in the remaining wells. Columns seven through 11 rows E through G.Do not remove any media, but instead, add 100 microliters of cell titer glow reagent to obtain a one-to-one dilution in a total volume of 200 microliters. Immediately place the plate onto a mutating or orbital shaker for 10 minutes to mix the samples while they incubate.
Then transfer 60 microliters from each well into a white walled 96. Well plate luminescence values are higher in white plates than clear or black plates because they reflect light upwards towards the detector. If any bubbles form during the transfer, pop them using forced air from a plastic transfer pipette bulb.
Then within 12 minutes of the addition of the reagents, place the plate on a luminometer and read the luminescence values. Average the luminescent values from the three or six wells in each group and plot as a function of cell number proceed with the highest dilution of reagents that still give linear and proportional results as defined in the accompanying text. This will save on cost for the infrared assays, harvest cells, and reflate them at different densities as described in the accompanying text protocol.
Fill the surrounding wells with 200 microliters of sterile water to reduce the effect of temperature gradients and evaporation along the edges. Let the cells adhere overnight at 37 degrees Celsius in a CO2 buffered incubator, and then fix the cells by adding 100 microliters of a fixative, such as 4%formaldehyde and 4%sucrose in 0.1 molar phosphate buffer. Incubate the plate, add room temperature for 20 minutes, then remove the fixative and wash the cells three times with 200 microliters of PBS.
Next, dilute the blocking solution one-to-one in PBS and add 0.3%Triton X 100 as a cell permeable. Then add 35 microliters of the blocking solution to each well taking care to avoid forming bubbles in the wells. Incubate the plate with the blocking solution for 30 to 60 minutes at room temperature.
During this incubation, dilute the primary antibodies for the proteins of interest. For example, dilute the Antifa tubulin primary antibody, one to 10, 000 in a one-to-one mixture of blocking buffer and PBS with 0.3%TRITTON X 100 while optimizing the procedure test. Additional antibody dilutions here, the Antifa tubulin primary antibody is also diluted one to 5, 000 in a one-to-one mixture of blocking buffer and PBS with 0.3%Triton X 100.
Next, add the antibodies at the concentrations shown here to the cells fixed on the plate. Be sure to leave the negative control wells in column two in the blocking solution as they will only be exposed to secondary antibodies. Incubate the cells in the primary antibodies for one to two hours at room temperature or overnight at four degrees Celsius.
Next, prepare the 800 nanometer anti mouse IgG secondary antibodies by diluting them one to 1000 or one to 2000 in a one to one mixture of blocking buffer and PBS with 0.3%Triton X 100 following primary antibody incubation. Wash the wells three times for 10 minutes each with 200 microliters of PBS per. Well then add 35 microliters of secondary antibodies to the wells.
Add the one to 1000 dilution to the top half of the plate and the one to 2000 dilution to the bottom half. Incubate the cells in the secondary antibodies for one hour at room temperature while protected from light. Then wash off the unbound secondary antibodies with three 10 minute washes of PBS using 200 microliters per well each time.
Next, dilute the DRAC five stock one to 10, 000 for a final concentration of 0.5 micromolar and the sapphire stock, one to 1000 in PBS with 0.3%Triton X 100. Add 35 microliters of this mixture to the left half of the plate, but do not add anything to the negative control wells in column two. For the right half of the plate, add 35 microliters of drac five diluted one to 20, 000 and sapphire diluted one to 2000.
Incubate in these solutions for 30 minutes at room temperature away from light. Then wash the plate three times with 200 microliters of PBS per well for 10 minutes each. Begin by scanning the plates on the infrared imager at intensity five and a resolution of 169 microns.
Either medium quality or low quality settings are sufficient. Then scan at different focus offsets of 2.5 millimeters, 3.0 millimeters, 3.5 millimeters, and 4.0 millimeters to see where the highest signal to noise ratio and crispus signal is achieved. Once the focus offset is optimized, use the incel western function in the software to place the right sized grid onto the image of the plate and export the data into Microsoft Excel.
Subtract the average of the integrated intensities in the negative control wells from every corresponding data point in the 700 and 800 nanometer channels. Then average the integrated signal intensities for each group of wells and plot the data as a scatter plot against cell density after optimization. The signal strength in all three assays was significantly correlated with the number of cells per well, although all three assays were highly linear, they were not equivalent in sensitivity.
The A TP assay showed the highest sensitivity of the three defined as the most proportional change of luminescent output with reductions in cell number in rat primary neuronal cells. The optimal reagent concentrations were different and the assays exhibited less linearity throughout the entire range of the assay. However, the assays were both linear and sensitive at plating densities below 100, 000 cells shown here.
Our dose response curves of neuroblastoma cells treated with MG 1 32 a proteasome inhibitor MG 1 32 was applied in the presence or absence of the glutathione precursor and acetyl cysteine N acetyl cysteine shifts all three curves to the right. For example, the IC 50 as measured by DRAC five plus sapphire was 1.64 micromolar MG 1 32 without n-acetylcysteine and 4.64 micromolar mg 1 32 in the presence of N-acetylcysteine. This, this was similar to the values found in the alpha tubulin assay of 1.96 micromolar and 6.35 micromolar of MG 1 32 respectively.
However, a TP levels were raised at low concentrations of MG 1 32, a phenomenon called hormesis. These data reveal that a TP levels are not necessarily in proportion to cell titer upon treatment, and that N-Acetyl cysteine can also protect metabolic function. Once mastered the luminescent, A TP assay can be completed within 30 minutes, and the infrared staining can be easily completed within eight hours.
While attempting these procedures, it's important to remember that they do not measure cell numbers in the same way as a high resolution microscope. For example, one caveat is that alpha tubulin protein levels or a TP levels may be changed by the treatments in the absence of parallel changes in cell numbers.
