Evaluating Autophagy Levels in Two Different Pancreatic Cell Models Using LC3 Immunofluorescence

The goal of this protocol is to determine autophagic levels in pancreatic cancer and pancreatic acinar cells through LC3 immunofluorescence and LC3 dot quantification.

The LC3 immunofluorescence enables the determination of autophagy levels in pancreatic cells. It permits the study of the potential effects of different agents on pancreatic cell autophagy. Immunofluorescence is a technique utilized to detect endogenous LC3 protein, which avoids problems caused by over-expressing LC3, such as forming protein aggregates unrelated to autophagy.

Demonstrating the procedure will be Felipe Renna, a PhD graduate, and Malena Herrera Lopez, a PhD student from the laboratory of Maria Ines Vaccaro. To begin cell preparation, soak 12-millimeter round cover slips in absolute ethanol. After removing the cover, place the soaked cover slip vertically in the 24-well plate.

Expose the multi-well plate to ultraviolet radiation for 15 minutes. Then position the cover slip horizontally and wash it with DMEM. Now see the low passage number of pancreatic cells, re-suspended in DMEM, containing 10%FBS, penicillin, and streptomycin, and incubate the cells for two days.

Two days after cell seeding, prepare gemcitabine solution in DMEM at one microgram per microliter concentration. Then aspirate half the volume from each well in a separate tube, and add appropriate amount of gemcitabine solution. Return the half volume corresponding to each well back into the treated wells, and incubate the cells for 24 hours.

For fixing and permeabilizing the cells, add cold methanol into a 24-well plate containing cells. Also prepare a six-well plate with cold PBS. Using a syringe needle and tweezers, take each cover slip and wash it twice in PBS.

Then incubate in methanol for six minutes. After washing twice with PBS, incubate the cover slips in a blocking solution for one hour. Add an anti LC3 in the blocking solution at a ratio of one to 1000, and maintain on ice.

Place a piece of laboratory sealing film over the multi-well lid, and add 25 microliters of anti-LC3 solution per cover slip over the sealing film. Using a syringe needle and tweezers, place each cover slip over the primary antibody drop, ensuring the cell side is in contact with the solution. Place the multi-well plate into the humidity chamber.

Cover it with foil and incubate overnight in the fridge. The next day, remove the multi-well plate from the humidity chamber. Place the cover slips back on the multi-well plate and wash them with PBS three times.

Dilute fluorescently-labeled anti-rabbit in blocking solution at a ratio of one to 800, and keep on ice in the dark. After sealing the multi-well lid, add 25 microliters of anti-rabbit solution per cover slip over the sealing film. And treat the cover slip with a secondary antibody as previously demonstrated.

Then incubate the plate in the humidity chamber for two hours at room temperature, protected from light. At the end of incubation with antibody, wash the cover slips on the multi-well plate with PBS three times. Incubate each cover slip with the prepared DAPI solution for 10 minutes.

Once washed with PBS, maintain the multi-well plate in the dark. For cell montage, prepare two beakers with water and a piece of paper. Add 10 microliters of PVA-DABCO solution per cover slip on a slide.

Wash the cover slip in water, and then dry it on paper. Finally, place it over the PVA-DABCO drop. And dry overnight in the dark.

The next day, using an inverted confocal microscope, visualize the labeled cells and capture the images. After capturing images, drag and drop each image file containing the captured channels in the Fiji screen. In the dialogue box, click OK to open the image.

Then close the opened console window. From the Image tab, select Color, and then Split Channels. Close the image corresponding to the channels other than the LC3 image.

Then in the Image tab, select Adjust, followed by Color Balance. Move the maximum slider to the left until the image saturates to visualize the cell contours. Use the freehand selection tool to draw the cell outline, and click reset to adjust the colors.

To cut the selected item from the Edit tab, select Cut, and close the image without saving it. And from the Edit tab, select paste. In the Analyze menu, choose the tool 3D Objects Counter.

Set the threshold at 2000, and the size filter at 50 to 500. Ensure the objects and summary boxes are marked, and click on OK.In the log window, the number of dots will be described as objects detected. The immunofluorescence indicated the cellular distribution of LC3 protein, whereas the DAPI showed nuclear localization in pancreatic cells.

The LC3 dots significantly increased under gemcitabine treatment, indicating autophagic activity. Under an excessive confluence, the exocrine pancreas cells tend to pile up and grow on top of each other. The paraformaldehyde cell fixation method was ineffective for preserving LC3 proteins, as it did not reflect accurate distribution.

When fixation or treatment was performed without waiting two days after seeding, the PANC1 cells had a round shape. This morphology decreased the relation between the cytoplasm and nucleus, making it difficult to understand the intracellular distribution of LC3. Incomplete methanol fixation could interfere with proper LC3 immuno labeling, leading to unclear images and suboptimal quantification.

The most important thing to remember during this procedure is that the cells must be fixed in cold methanol for six minutes, instead of paraformaldehyde. Following this procedure, colocalization between LC3 and other proteins can be analyzed, allowing the study of different molecular pathways related to LC3 functions.