This protocol allows for quick and easy detection of primary cilia in cultured cells, within a variety of research endeavors. The method could be utilized in disorder that affect the basal bodies of primary cilia. This procedure can also be applied for staining primary cilia in paraffin embedded tissue or for other experiments where fluorescence is needed.
Begin by autoclaving 22 by 22 millimeter cover slips, and preparing materials for the experiment. Thaw FBS and penicillin streptomycin, and warm the culture medium to room temperature. Passage the cells with trypsin EDTA and PBS.
Prepare fresh 4%paraformaldehyde, culture medium, and 1%gelatin solution, according to manuscript directions. Then clean the laminar flow hood with 70%ethanol, and place all required materials inside. Paraformaldehyde is extremely hazardous.
Proper PPE has to be worn at all times, and it should only be handled in a chemical hood. After growing the desired cells to 70%confluence remove them from the incubator, and place them in the laminar flow hood. Remove the culture medium and rinse the cells twice with PBS.
Add about two milliliters of 0.25%trypsin EDTA to the culture flask, and incubate it at 37 degrees Celsius for five minutes. Check the cells periodically on the inverted microscope to monitor detachment. When the cells have detached gently resuspend them in 10 milliliters of culture medium, pipetting carefully to create a single cell suspension.
Transfer the cell suspension to a 50 milliliter conical tube and centrifuge it at 200 times G'for five minutes. Decant the supernatant then add 10 milliliters of culture medium, and gently resuspend the pellet. Take 20 microliters of the cell suspension and mix it with trypan blue at a one-to-one volume ratio.
Then count the cells using standard hemocytometry. Place a cover slip inside each well of a six well plate, and coat the cover slip with two milliliters of the gelatin solution, which will help the cells attached to the cover slip. Remove the gelatin and let the plate air-dry for a few minutes.
Seed 100, 000 fibroblasts into each well and add two milliliters of culture medium. Then incubate the cells for 24 hours at 37 degrees Celsius, five percent carbon dioxide, and 90%relative humidity. After the incubation treat the cells to induce ciliation.
Warm the 4%paraformaldehyde to room temperature, and prepare a pasture pipettes, ABS, a waste container, 15 milliliter conical tubes, micro pipettes, and tips. Take the cells from the incubator, and place them on the lab bench. Remove media from each well, leaving the cover slip.
Gently wash the cells three times with two milliliters of PBS. Then add two milliliters of the 4%PFA into each well to fix the cells. Incubate the plate for 10 minutes at room temperature, then remove the PFA, and repeat the washes with PBS.
Add two milliliters of 0.5%Triton X-100 to each well, and incubate the plate for 15 minutes. Then wash the wells four times with PBS. To make the blocking solution, thaw a goat serum for five minutes, and dilute it in PBS at a one to 20 ratio.
Add 150 microliters of this solution to each cover slip, and incubate the plate for 20 minutes. Thaw the primary antibodies for five minutes, and dilute them separately in PBS, as described in the text manuscript. Remove the blocking solution from the cover slips and add 150 microliters of both antibody solutions.
Then incubate the plate for 60 minutes at room temperature. Remove the primary antibodies and gently wash the cover slips three times with two milliliters of PBS. Dilute Cy3 Sheep Anti-Mouse and Alexa fluor 488 Goat anti-Rabbit in PBS, at a one to 300 ratio, and add 150 microliters of both secondary antibody solutions to the cover slip.
Prepare a working DAPI solution by diluting 10 microliters of the stock in 50 milliliters of PBS, and add two milliliters of this dilution to the cover slips. Incubate the plate for five minutes in the dark, at room temperature. Before placing the cover slips on the slides, prepare two needles, tweezers, and mounting media, and label all the slides.
Remove the DAPI solution from the wells, and wash them three times with PBS. Put one drop of mounting media on each slide. Use the needle to gently lift the cover slip from the well's bottom.
Then flip it and gently place it over the drop of mounting media using the tweezers. Carefully remove any bubbles. Protect the slides from light and store them overnight at four degrees Celsius.
On the next day use a fluorescent or confocal microscope with a high magnification to visualize the primary cilia. This protocol was used to fix immunostain, and image various cell lines expressing primary cilia. When mouse myoblasts were exposed to ionizing radiation at various doses, it was found that higher doses induce the appearance of multiple primary cilia.
While low doses resulted in the appearance of single primary cilia. Similarly, when human lung fibroblasts were radiated at a low dose, single primary cilia were detected by immunofluorescence. An increase in primary cilia was observed in mouse embryonic fibroblasts that were starved, demonstrating that metabolic stress affects the incidents of primary cilia.
When skin fibroblasts were treated with 120 nanomolar doxorubicin, they expressed a single primary cilium. Higher doses induce the appearance of multiple primary cilia. Fibroblasts treated with 1.25 nanomolar Taxol also expressed a single primary cilium.
But multiple cilia were not detected after treatment with higher doses. When opening this protocol make sure to follow all PPE regulations. Keep in mind the culture needs of your cell line of choice and chose your antibodies carefully.
This procedure cannot be directly followed by other procedures, since the cells are irretrievable. Instead, parallel experiments should be programmed.
