Cell culture and calcium switch assay

Calcium Switch Assay for Synchronized Junction Disassembly and Reassembly in Human Intestinal Epithelial Monolayers

Arturo Raya-Sandino and Asma Nusrat*

Department of Pathology, University of Michigan Medical School, Ann Arbor, MI, USA

For correspondence: anusrat@umich.edu

Abstract

The calcium switch assay provides a synchronized system for examining the disassembly and reassembly of calcium-dependent epithelial cell-cell junctions^1-3. Here, we describe a protocol for evaluating the recruitment of the desmosomal cadherin desmoglein-2 (DSG2) and the adherens junction protein E-cadherin to cell-cell contacts in human intestinal epithelial monolayers. Pre-established non-targeting control and desmocollin-2 (DSC2)-knockdown SKCO15 cell populations are exposed to serum-free low-calcium (1 µM) medium to disrupt intercellular junctions, followed by calcium restoration (1.8 mM) to initiate synchronized junction reassembly. Barrier recovery is monitored by sequential transepithelial electrical resistance (TEER) measurements, while the temporal recruitment of DSG2 and E-cadherin to cell-cell borders is evaluated by immunofluorescence labeling and confocal microscopy. This approach enables comparison of desmosomal and adherens junction reassembly under control and DSC2-deficient conditions⁴.

Keywords: Desmosome Assembly, Calcium Switch Assay, Epithelial Barrier, TEER.

Materials and Reagents

1. Pre-established non-targeting control and DSC2-knockdown SK-CO15 human intestinal epithelial cells. Parental SK-CO15 cells were provided by Dr. Enrique Rodriguez-Boulan (Weill Cornell Medicine; RRID: CVCL_A337). Generation and validation of the control and DSC2-knockdown populations are described in references^4-6.

2. Calcium-containing complete growth medium: DMEM, 1× (Gibco, catalog number 11966-025), supplemented with 10% (v/v) FBS (Gibco, catalog number 10082-147) and 1% (v/v) penicillin-streptomycin (Gibco, catalog number 15140-122).

3. DMEM formulated without calcium chloride (Gibco, catalog number 21068-028).

4. Calcium- and magnesium-free PBS (PBS⁻; Corning, catalog number 21-040-CV).

5. Calcium- and magnesium-containing DPBS (PBS⁺; Corning, catalog number 21-030-CV).

6. CaCl₂, 1 M stock solution (Sigma-Aldrich/MilliporeSigma, catalog number C-34006).

7. CaCl₂ working solution: Dilute the 1 M CaCl₂ stock 1:1,000 in sterile molecular biology-grade water to prepare a sterile 1 mM working solution. Dilute this working solution 1:1,000 in DMEM formulated without calcium chloride to obtain serum-free low-calcium medium containing a nominal concentration of 1 μM added CaCl₂.

8. Molecular biology-grade water (Corning, catalog number 46-000-CV).

9. Transwell permeable supports with 3 μm pore-size polycarbonate membranes, 24-well format, 6.5 mm insert diameter, and 0.33 cm² growth area (Corning Costar, catalog number 3472).

10. EVOM epithelial voltohmmeter equipped with a chopstick electrode compatible with 24-well Transwell inserts (World Precision Instruments), used to confirm monolayer formation before the calcium switch.

Controls

1. Non-targeting control: Use a pre-established SKCO15 cell population expressing a non-targeting control shRNA. Subject these cells to the same culture, calcium-switch, TEER, fixation, and staining procedures as the DSC2-knockdown cells.

2. DSC2-knockdown condition: Use a pre-established SKCO15 cell population with stable DSC2 depletion. Verify that DSC2 depletion is maintained before performing the calcium-switch assay, for example by immunoblotting or immunofluorescence microscopy.

3. Non-treated control: Maintain parallel non-targeting control and DSC2-knockdown monolayers continuously in complete growth medium. These samples define steady-state TEER and junctional DSG2 and E-cadherin localization.

4. Low-calcium endpoint control: Collect monolayers immediately after the 20 h incubation in serum-free low-calcium medium and before calcium restoration. This condition verifies the disruption of calcium-dependent junctions and establishes the starting point for junction reassembly.

5. Cell-free blank inserts for TEER: Include cell-free inserts containing the same medium as the corresponding experimental samples. Use separate blanks for serum-free low-calcium medium and complete growth medium because the media may have different conductivities.

6. Immunofluorescence controls: Include no-primary-antibody or secondary-antibody-only controls to evaluate nonspecific secondary-antibody binding, background fluorescence, and membrane autofluorescence.

7. Comparison with E-cadherin dynamics: Concurrent analysis of E-cadherin helps determine whether DSC2 depletion selectively affects DSG2 recruitment or produces a wider defect in junction reassembly. However, unchanged E-cadherin localization should not be interpreted as proof that all intracellular trafficking mechanisms remain intact.

Experimental Design

A. Establishment of epithelial monolayers on Transwell supports

1. Seed 1 x 10⁵ pre-established non-targeting control or DSC2-knockdown SKCO15 cells per insert in 250 μL of complete growth medium in the apical compartment.

2. Add 600 μL of complete growth medium to the basolateral compartment.

3. Culture the cells at 37 °C and 5% CO₂, replacing the medium in both compartments every 2 days.

4. Monitor monolayer formation by phase-contrast microscopy and transepithelial electrical resistance (TEER). Initiate the calcium-switch assay when the monolayers reach a stable TEER of approximately 500 Ω·cm².

B. Calcium depletion and synchronized junction reassembly

1. Prepare serum-free low-calcium medium consisting of DMEM formulated without calcium chloride and supplemented with 1 μM added CaCl₂.
2. Prepare complete growth medium for calcium restoration.
3. Warm both media to 37 °C before use.
4. Reserve parallel non-targeting control and DSC2-knockdown inserts as unswitched controls. Maintain these inserts continuously in complete growth medium.
5. Aspirate the complete growth medium from the apical and basolateral compartments of the inserts assigned to the calcium-switch assay.
6. Gently wash both compartments twice with prewarmed calcium- and magnesium-free PBS (PBS-). Avoid directing the liquid stream onto the cell monolayer.
7. Remove the PBS- completely.
8. Add 250 μL of serum-free low-calcium medium to the apical compartment.
9. Add 600 μL of serum-free low-calcium medium to the basolateral compartment.
10. Incubate the monolayers for 20 h at 37 °C and 5% CO₂ to disrupt calcium-dependent intercellular junctions.
11. At the end of the low-calcium incubation:

-Measure TEER using a cell-free blank insert containing the same serum-free low-calcium medium, if this measurement is included in the experimental design; and fix separate replicate inserts to document the low-calcium endpoint.

1. Aspirate the low-calcium medium from both compartments.
2. Add 250 μL of prewarmed complete growth medium to the apical compartment and 600 μL to the basolateral compartment.
3. Define the addition of complete growth medium as (t=0) for synchronized junction reassembly.
4. Measure TEER at predefined intervals, such as 0, 1, 2, 4, 8, and 24 h after calcium restoration.
5. At each time point, fix separate replicate inserts for immunofluorescence analysis.

References

1          Cereijido, M. et al. The making of a tight junction. J Cell Sci Suppl 17, 127-132 (1993). https://doi.org:10.1242/jcs.1993.supplement_17.18

2          Martinez-Palomo, A., Meza, I., Beaty, G. & Cereijido, M. Experimental modulation of occluding junctions in a cultured transporting epithelium. J Cell Biol 87, 736-745 (1980). https://doi.org:10.1083/jcb.87.3.736

3          Gonzalez-Mariscal, L., Chavez de Ramirez, B. & Cereijido, M. Tight junction formation in cultured epithelial cells (MDCK). J Membr Biol 86, 113-125 (1985). https://doi.org:10.1007/BF01870778

4          Raya-Sandino, A. et al. Regulation of intestinal epithelial intercellular adhesion and barrier function by desmosomal cadherin desmocollin-2. Mol Biol Cell 32, 753-768 (2021). https://doi.org:10.1091/mbc.E20-12-0775

5          Flemming, S. et al. Desmocollin-2 promotes intestinal mucosal repair by controlling integrin-dependent cell adhesion and migration. Mol Biol Cell 31, 407-418 (2020). https://doi.org:10.1091/mbc.E19-12-0692

6          Kolegraff, K., Nava, P., Helms, M. N., Parkos, C. A. & Nusrat, A. Loss of desmocollin-2 confers a tumorigenic phenotype to colonic epithelial cells through activation of Akt/beta-catenin signaling. Mol Biol Cell 22, 1121-1134 (2011). https://doi.org:10.1091/mbc.E10-10-0845