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Fixing and staining tissue culture cells or slices grown in a 3D collagen matrix with fluorescent phalloidin

A. Fluorescent Phalloidin Staining Protocol

B. Preparation of 3D cultures

C. Phalloidin staining of slice cultures grown on 3D collagen matrices.

Cells or tissue slices are grown in 3D collagen matrices to more accurately replicate the in vivo cellular milieu that cells function in to study how cell morphology and function respond to the extracellular environment confining the cell. To faithfully capture the integrity and structure of the F-actin cytoskeleton (and any changes it undergoes) with a fluorescent phalloidin stain, proper fixation and handling of the cells/slices is necessary.  For fixation of cells for phalloidin staining, methanol must be avoided and instead, paraformaldehyde or glutaraldehyde should be used as they provide excellent actin filament staining and good lamellipodia preservation.

A. Fluorescent Phalloidin Staining Protocol

Reagents

  • Semi-confluent 3D tissue culture cells or tissue slices (see supplemental details below for preparing these cultures)
  • Acti-stain 488 phalloidin (Cat. # PHDG1).  Other options include Acti-stain 535 (Cat. # PHDR1), Acti-stain 555 (Cat. # PHDH1), and Acti-stain 670 (Cat. # PHDN1)
  • Either obtain the F-actin staining kit from Cytoskeleton, Inc. (Cat. # BK005) or prepare reagents below
  • Fixative solution (3.7% paraformaldehyde in PBS, pH to 7.0 is necessary)
  • Permeabilization buffer (0.5 % Triton X-100 in PBS)
  • Antifade mounting medium (Fluka BioChemika, Cat. # 10981)
  • Coverslip sealing solution (clear nail polish)

 

Equipment

    • Fluorescent microscope with FITC excitation filter at 450-480 +/- 20 nm and emission filter at 535 +/- 20 nm for Acti-stain™ 488. For Acti-stain red fluorophores, the excitation filter is 535 +/- 20 nm and emission filter at 585 +/- 20 nm for Acti-stain 535 and 555.  For Acti-stain 670, the excitation filter is at 630 +/- 20 nm and the emission filter is at 680 +/- 20 nm.
    • Digital CCD camera.

     

    Method

    Phalloidin Staining Protocol

    1. Reconstitute phalloidin according to manufacturer’s directions.  Dilute to working concentration as directed in PBS. Keep working stock of fluorescent phalloidin in the dark at room temperature.
    2. Fix cells in the collagen gels with 3.7% (v/v) paraformaldehyde 10 min at room temperature.
    3. Rinse 3 times in PBS.
    4. Permeabilize cells with 0.1 -0.5% (v/v) Triton X-100 for 10 min at room temperature.
    5. Rinse 3 times in PBS.
    6. Cut collagen gels into ~1 mm3 samples.
    7. Stain with 0.165 μM Acti-stain phalloidin for 30 min in the dark at room temperature.  Note: This concentration may need to be empirically determined.  Cell nuclei can be stained with 100 nM DAPI in PBS in concert with phalloidin if desired.
    8. Wash 3 times with PBS.
    9. Place stained samples on glass slides and gently press with a glass coverslip using a drop of anti-fade mounting medium to prevent photo-bleaching.
    10. If possible, seal each side of coverslip with nail polish.
    11. Either view immediately or store the slides in the dark at 4°C for up to 24 hrs.
    12. Visualize the cells in stained samples with an appropriate fluorescence microscope set-up. For example, a confocal fluorescent microscope with a 63X oil-immersion lenses at the appropriate filter excitation and emission settings.
    13. For analysis of staining in 3D gels, collection of 5-10 representative maximal projection z-stacks from each sample are useful to determine the average brightness of cells within each image.

    B. Preparation of 3D cultures

    Prepare collagen-coated polyacrylamide gel substrate for cells

    • 25 mm and 18 mm diameter coverslips (ThermoFisher or VWR)
    • Acrylamide powder (ThermoFisher or VWR)
    • Bis-acrylamide powder (ThermoFisher or VWR)
    • Collagen, type 1 solution from rat tail (Sigma, Cat. # C3867)
    • 6 well culture plates (ThermoFisher or VWR)
    • Phosphate-buffered saline (PBS, 50 mM potassium phosphate pH 7.4, 50 mM NaCl)
    • Sulfo-SANPAH (sulfosuccinimidyl 6-(4'-azido-2'-nitrophenylamino) hexanoate (ThermoFisher, Cat. # 22589)

     

    3D Culture Method

    1. Grow tissue culture cells in appropriate growth medium and maintain at 37°C in a humidified, 5% CO2 environment.
    2. Prepare chemically-modified glass coverslips in sets (25 mm diameter and 18 mm diameter). Larger size coverslips can be used. See references for chemical treatment protocol.
    3. Prepare polyacrylamide solution. Note: the stiffness of the gel can be adjusted by the percentage of acrylamide gel produced. Substrate stiffness is an essential parameter to monitor as it has a direct impact on cell morphology and function.  Different cell types have different optimal substrate stiffness.  Consult the literature for the particular cell type to be used.
    4. Pipette a fixed volume of acrylamide solution onto a 25 mm diameter coverslip and place the 18 mm diameter coverslip on top of the acrylamide solution.
    5. Allow polymerization to occur (10 min) and then remove the top coverslip.
    6. Place the bottom coverslip with the attached polyacrylamide gel into a multi-well plate (6 well plate is typically used) with PBS solution. 
    7. The surface of the gel is cross-linked with type I rat tail collagen using sulfo-SANPAH for 4 hrs at 4°C. Collagen concentration will need to be determined empirically. Typical concentrations range from 1-3 mg/ml.
    8. Polymerize collagen at 37°C, 5% CO2 and 95% humidity for 90 min.
    9. Wash collagen matrices 3 times with PBS to remove uncross-linked collagen.
    10. Seed cells onto collagen-coated polyacrylamide gels and continue to grow and experimentally treat under cell culture conditions as chosen by experimenter. Migrating cells will move away from the polyacrylamide gel substrate.

     

    For detailed information on these reagents and methods, please see

    • Yeung T. et al. 2005. Effects of substrate stiffness on cell morphology, cytoskeletal structure, and adhesion. Cell Motil. Cytoskeleton. 60, 24-34.
    • Byfield et al. 2009. Endothelial actin and cell stiffness is modulated by substrate stiffness in 2D and 3D J. Biomech. 42, 1114–1119.
    • Wang Y.-L. and Pelham, R.J. Jr. 1998. Preparation of a flexible porous polyacrylamide substrate for mechanical studies of cultured cells. Methods Enzymol. 298, 489–496.
    • Many more citations can be found by searching Google Scholar with the search terms “phalloidin 3D collagen”.

    C. Phalloidin staining of slice cultures grown on 3D collagen matrices.

    Reagents and Equipment:

    Same as Protocol 3 above with the following additions/exceptions

    • CB buffer (10 mM MES pH 6.1, 138 mM KCl, 3 mM MgCl2, 2 mM EGTA)
    • NaBH4 (sodium borohydride)
    • Tissue slices rather than tissue culture cells

     

    Method:

    1. Prepare collagen-coated polyacrylamide gels as described above.
    2. Cross-link 1-3 mg/ml collagen to the polyacrylamide gel for 4 hrs at 4°C using sulfo-SANPAH.
    3. Polymerize collagen at 37°C in a humidified chamber (37°C, 5% CO2 and 95% humidity) for at least 6 hrs followed by multiple rinsings with warm medium to remove uncross-linked collagen. 
    4. Prepare tissue slice cultures from the region of interest of control and experimental animals.
    5. Place slices on the collagen matrix, positioning cells of interest toward the collagen if applicable.
    6. Secure each tissue slice to the collagen matrix with either dialysis tubing or by polymerizing 1.6 mg/ml rat tail collagen I (in medium) on top of the slice.
    7. Grow and experimentally treat under tissue culture conditions as chosen by experimenter. Migrating cells will move away from the polyacrylamide gel substrate.

     

    Phalloidin Staining Protocol:

    1. Reconstitute phalloidin according to manufacturer’s directions.  Dilute to working concentration as directed. Keep working stock of fluorescent phalloidin in the dark at room temperature.
    2. Remove collagen matrices containing cells from dishes, cut into smaller pieces, and place into 12 well dishes.
    3. Pieces of matrices are fixed in 4% paraformaldehyde (CB buffer containing 0.1% Triton X-100) for 30 min at room temperature.
    4. Rinse fixed tissue with CB buffer + 0.2% Triton X-100 for 30 min at room temperature.
    5. Optional: Reduce collagen matrix autofluorescence with 2 x 10 min washes in CB buffer + 0.5 mg/ml NaBH4 (sodium borohydride) at room temperature.
    6. Block fixed and permeabilized gels in CB buffer + 2% BSA, 1% goat serum, 0.2% Triton X-100 overnight at 4°C.
    7. If applicable, matrix pieces are then incubated with a primary antibody of interest overnight in blocking buffer and then rinsed in CB buffer + 0.2% Triton X-100 multiple times at room temperature.
    8. After primary antibody incubation (if performed), fluorescent secondary antibody is added for a 4-6 hrs incubation at 4°C.
    9. In place of primary and secondary antibodies, or in parallel with the secondary antibody, 0.165 mM phalloidin can be used to stain F-actin (4-6 hrs incubation at 4°C).  The phalloidin concentration may need to be determined empirically.
    10. Matrix pieces are rinsed overnight at 4°C in CB buffer.
    11. Mount matrix piece on glass slide and gently press with a glass coverslip using a drop of anti-fade mounting medium to prevent photo-bleaching.
    12. If possible, seal each side of coverslip with nail polish.
    13. Either view immediately or store the slides in the dark at 4°C for up to 24 hrs.
    14. Visualize the cells in stained samples with an appropriate fluorescence microscope set-up. For example, a confocal fluorescent microscope with a 63X oil-immersion lenses at the appropriate filter excitation and emission settings.
    15. For analysis of staining in 3D gels, collection of 5-10 representative maximal projection z-stacks from each sample are useful to determine the average brightness of cells within each image.

     

    For detailed information on these reagents and methods, please see:

    • Fischer R.S. et al. 2009. Local cortical tension by myosin II guides 3D endothelial cell branching. Curr. Biol. 19, 260–265.
    • Lo C.-M. et al. 2000. Cell movement is guided by the rigidity of the substrate. Biophys. J. 79, 144-152.
    • Wang Y.-L. and Pelham, R.J. Jr. 1998. Preparation of a flexible porous polyacrylamide substrate for mechanical studies of cultured cells. Methods Enzymol. 298, 489–496.