Simple Detection of Primary Cilia by Immunofluorescence

Summary

Primary cilia are extracellular structures associated with the centriole. Primary cilia detection by immunofluorescent staining is a relatively simple procedure that results in extremely high-quality images. In this protocol, fibroblasts expressing primary cilia were fixed, immunostained, and imaged in a fluorescent or confocal microscope.

Abstract

Primary cilia are dynamically regulated during cell cycle progression, specifically during the G0/G1 phases of the cell cycle, being resorbed prior to mitosis. Primary cilia can be visualized with highly sophisticated methods, including transmission electron microscopy, 3D imaging, or using software for the automatic detection of primary cilia. However, immunofluorescent staining of primary cilia is needed to perform these methods. This publication describes a protocol for the easy detection of primary cilia in vitro by staining acetylated alpha tubulin (axoneme) and gamma tubulin (basal body). This immunofluorescent staining protocol is relatively simple and results in high-quality images. The present protocol describes how four cell lines (C2C12, MEF, NHLF, and skin fibroblasts) expressing primary cilia were fixed, immunostained, and imaged with a fluorescent or confocal microscope.

Introduction

Primary cilia are sensory, solitary, membrane-bound, nonmotile structures associated with the cell’s mother centriole. Primary cilia are found on most vertebrate cells with the exception of red blood cells, adipocytes¹, and hepatocytes². Primary cilia are formed as an elongated axoneme composed by microtubules, whose main component is α-tubulin. The axoneme grows from the basal body, which is structured from γ-tubulin. The length of the primary cilia varies between 2–10 µm; however, its dimensions can change during glycylation, starvation, hypoxia, cytotoxic stress, or after exposure to ionizing radiation^3,4,5,6,7. Usually, cells have only one primary cilium, which is involved in morphogenesis and cell signalling pathways important for cell proliferation and differentiation^8,9.

Primary cilia are dynamically regulated during cell cycle progression, specifically during the G0/G1 phases, and resorbed before entering mitosis in a process associated with tubulin deacetylation mediated by HDAC6 (histone deacetylase 6)¹⁰. The exact moment of primary cilia resorption depends upon cell type and the expression of genes directly involved in this process, such as Aurora A, Plk1, TcTex-1^11,12,13. Depending on the cell type, the primary cilia express different types of receptors, ion channels, and active signalling pathways. These include the most important signalling receptors affecting proliferation and survival, EGFR, PDGFR, and FGFR. Also included are some of the signalling pathways that may affect the function of one or more organs, including Hedgehog, Notch, and Wnt. Thanks to these receptors and signalling pathways, the primary cilia also perform a chemosensory function. This function allows primary cilia to detect specific ligands for Notch, hormones, and biologically active substances such as serotonin or somatostatin. Other specific functions exhibited by primary cilia of different lengths include reaction to changes in temperature, gravity, and osmolality¹⁴.

Primary cilia can be visualized through various methods, such as live visualization, transmission electron microscopy, 3D imaging, or by software for the automatic detection of primary cilia^5,15,16,17. However, these methods are highly specialized and ongoing research needs basic, fast, and easy methods for staining primary cilia in every stage of research. Described is an easy and useful method for the detection of primary cilia in cultured cells.

Protocol

1. Preparation of culture media, solutions, and dishes

1. Autoclave the coverslips (22 x 22 mm). Prepare 6 well plates. Thaw fetal bovine serum (FBS) and antibiotic penicillin/streptomycin and warm the culture medium to room temperature (RT). Use trypsin-EDTA (0.25%) and 1x PBS (phosphate buffered saline with calcium and magnesium) to passage the cells.
2. Prepare fresh 4% paraformaldehyde (PFA) in dH₂O (800 mg of PFA in 20 mL of dH₂O). The PFA must be freshly prepared for each experiment. Stir and heat the solution at 55 °C for 30 min in the hood. Cool down at RT. Add 1 M sodium hydroxide until the solution becomes clear (pH = 7.2–7.4). Store at 4 °C for up to 1 week.
   Note: PFA is toxic; always wear adequate personal protective equipment and prepare in the chemical hood.
3. Prepare 500 mL of culture media, DMEM (Dulbecco´s Modified Eagle´s medium) containing 10% FBS, 1% penicillin/streptomycin, and 2% glutamine.
4. Prepare 13 mL of 1% gelatin solution in sterile dH₂O (130 mg of gelatin in 13 mL of dH₂O). Use 2 mL of 1% gelatin for each well in a 6 well plate. Keep sterile.
5. Clean the laminar flow hood using 70% ethanol. Place the required material inside the laminar flow hood before starting the experiment.

2. Cell culture for immunocytochemistry staining

1. Thaw the cells (in this study C2C12, MEF, NHLF, and skin fibroblasts) using standard techniques and plate them in a T75 flask supplemented with ~10–12 mL of the prepared media. Incubate at 37 °C/5% CO₂/90% relative humidity (RH) until the cells reach 70% confluence.
2. Remove the cells from the incubator and place them in the laminar flow hood. Remove the culture media and rinse the cells briefly 2x with 1x PBS. Add ~2 mL of 0.25% trypsin-EDTA into the T75 flask and incubate at 37 °C for ~5 min. Check periodically on the inverted microscope to monitor cell detachment.
   NOTE: The incubation time depends on the cell line and therefore must be determined empirically.
3. Gently resuspend the cells in 10 mL of culture media, pipetting carefully to create a single cell suspension. Rinse the flask again if necessary.
4. Place the cell suspension in a 50 mL conical tube and centrifuge for 5 min at ~200 x g. Decant the supernatant, add 10 mL of culture media, and gently resuspend the pellet. Take 20 µL of the cell suspension and mix in a 1:1 ratio with trypan blue and count in a cytometer following the standard method.
5. Place one coverslip inside each well of a 6 well plate using tweezers. Coat the coverslips with gelatin by pouring ~2 mL into the wells. This will help the cells attach to the coverslips. Remove the gelatin solution and let air-dry for a few minutes. The coverslips are now ready for cultivation of the cells. Start the cultivation immediately.
6. Seed 100,000 fibroblasts into each well and add 2 mL of culture media. Incubate the cells for 24 h at 37 °C/5% CO₂/90% RH. At this point, the cells can be treated according to the needs of the user. Treatments to induce ciliation have been previously described^4,5.
   NOTE: The initial seeding number depends on the cells’ doubling time and should be determined accordingly.

3. Immunofluorescent staining of primary cilia in vitro

1. Warm the 4% paraformaldehyde to RT. Prepare Pasteur pipettes, 1x PBS (RT), waste container, 15 mL conical tubes, micropipettes (0.5–10 µL, 20–200 µL, and 100–1,000 µL) and tips. Take the cells from the incubator and place them in the bench.
   NOTE: The staining procedure does not need to be performed in sterile conditions. All solutions must be at RT.
2. Remove media from each well. Leave the coverslip inside the well. Very gently wash the cells 3x with 2 mL of 1x PBS. Using a Pasteur pipette, add 2 mL of 4% PFA into each well to fix the cells. Incubate for 10 min at RT. Remove the PFA and wash 3x with 1x PBS.
   NOTE: Always use a sufficient volume to cover the entire coverslip during the incubation periods. Never let the cells dry. Never pour any of the solutions directly onto the coverslip.
3. Prepare 0.5% Triton X-100 in 13 mL of 1x PBS 10 min before use. Add 2 mL into each well. Incubate for 15 min. Wash gently 4x with 1x PBS.
   NOTE: Triton X-100 is insoluble in PBS at RT. Heat the 0.5% Triton X-100 solution to 37 °C in a water bath to dissolve it.
4. Thaw goat serum 5 min before use. Dilute the goat serum in 1x PBS in a 1:20 ratio as a blocking solution. Add 150 µL to each coverslip and incubate for 20 min at RT.
   NOTE: Prolong the blocking period up to 60 min if necessary. Do not wash the cells after blocking with goat serum.
5. Thaw the primary antibodies (i.e., anti-acetylated alpha tubulin and anti-gamma tubulin) 5 min before use. Dilute the antibodies separately in 1x PBS as follows: mouse anti-acetylated alpha tubulin in a 1:800 ratio and rabbit anti-gamma tubulin in a 1:300 ratio. Remove the blocking solution. Do not wash. Add 150 µL of both antibody dilutions to the coverslips and incubate for 60 min at RT.
   NOTE: If incubating overnight use 500–1,000 µL of the primary antibody solutions, seal the 6 well plate with paraffin film, and store at 4 °C. Alternatively, use 150 µL of antibody and incubate in a humidity chamber.
6. Remove the primary antibodies. Wash the coverslips very gently 3x with 2 mL of 1x PBS. Prepare the secondary antibodies in 1x PBS by separately diluting Cy3 sheep anti-mouse and Alexa Fluor488 goat anti-rabbit in a 1:300 ratio. Add 150 µL of both secondary antibody dilutions to the coverslips. Incubate for 45 min at RT in the dark.
   NOTE: Incubate in the dark to avoid photobleaching. Other combinations of secondary antibodies can be used as needed.
7. Prepare a DAPI (4', 6-diamidino-2-phenylindole) solution according to the manufacturer's instructions. Store the excess aliquots at -20 °C. Dilute 10 µL from a stock aliquot (1:5,000) in 50 mL of 1x PBS. Add 2 mL of this dilution to the coverslips. Incubate for 5 min at RT in the dark.
   NOTE: It is important to incubate the cells in the dark to avoid photobleaching. The DAPI dilution can be stored at 4 °C for up to 1 month.
8. Prepare 2 needles, slides, tweezers, and mounting media. Label the slides.
9. Remove the DAPI solution from the wells. Wash 3x with 1x PBS. Put one drop of mounting media on each slide. Use the needle to gently lift the coverslip from the well’s bottom. Flip the coverslip using the tweezers and gently place it over the drop of mounting media. Carefully remove any bubbles.
10. Protect the slides from light and store them overnight at 4 °C.
11. Use a fluorescent or confocal microscope with high magnification to visualize the primary cilia.
    NOTE: The slides can be stored in the dark at 4 °C for up to 2 months.

Results

The immunofluorescent staining of primary cilia is a relatively simple procedure that results in high-quality images. In these experiments, fibroblasts expressing primary cilia were fixed, immunostained, and imaged in a fluorescent or confocal microscope following the protocol described above. The primary cilium was detected using acetylated α-tubulin and γ-tubulin. The evaluation of primary cilia can be performed on various levels and any change in this regard can be linked to exposure to ionizing radiation, c...

Discussion

Several authors have described diverse methods for the detection of primary cilia, sometimes also describing various fixation methods that can affect their detection^6,20,21,22. Regardless, it is difficult to find a complete and straightforward protocol for detection. The ready availability of such a method would undoubtedly be of great assistance to the study of primary cilia investigation, esp...

Materials

Name	Company	Catalog Number	Comments
6-well plate	TPP	92406	Dimensions 128x86x22 mm
Alexa Fluor488	Jackson ImmunoResearch	111-546-047	AffiniPure F(ab')₂ Fragment Goat Anti-Rabbit IgG
Anti-Tubulin γ	Sigma-Aldrich	T5192	Polyclonal Rabbit anti-Mouse IgG2a
C2C12	ATCC	CRL-1772	Myoblast (mouse)
Cy3	Sigma-Aldrich	C2181	Anti-Mouse IgG (whole molecule) F(ab′)2 fragment–Cy3 antibody produced in sheep
Dapi (4′,6-Diamidino-2-phenylindole dihydrochloride)	Sigma-Aldrich	D9542
Dulbecco´s Modified Eagle´s medium	Thermo Scientific	11960044	High glucose, No glutamine, Gibco
Dulbecco’s Phosphate Buffered Saline	Sigma-Aldrich	D8662	With MgCl2 and CaCl2, Sterile-filtered, Suitable for cell culture
Fetal Bovine Serum	Thermo Scientific	16000044	Sterile-Filtered, Gibco
L-Glutamine	Sigma-Aldrich	G7513
MEF	ATCC	SCRC-1039	Mouse embryonic fibroblast
Monoclonal Anti-Acetylated Tubulin	Sigma-Aldrich	T7451	Monoclonal Anti-Acetylated Tubulin antibody produced in mouse
NHLF	Lonza	CC-2512	Primary lung fibroblasts (human)
Normal Goat Serum	Jackson ImmunoResearch	005-000-121
Paraformaldehyde	Sigma-Aldrich	158127-500G	Powder
Penicillin-Streptomycin	Sigma-Aldrich	P0781	10,000 units penicillin and 10 mg streptomycin per mL in 0.9% NaCl, Sterile-Filtered
ProLong Diamond Antifade Mountant	Thermo Scientific	P36961
Skin fibroblasts	Kindly gifted from Charles University, Faculty of Medicine in Hradec Králové.
Square Cover Slips	Thermo Scientific	22X22-1.5	Borosilicate glass, 22x22mm, Square
Triton X-100	Sigma-Aldrich	11332481001
Trypsin-EDTA (0.25%)	Thermo Scientific	25200072	Sterile-Filtered, Gibco