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Immunofluorescence staining of junctional proteins and adhesion molecules of hiPSC-derived EECM-BMEC-like cells on nanomembranes

Posted on by Pelin Kasap

I would like to share my successful immunofluorescence stainings of adhesion molecules and junctional proteins of hiPSC-derived brain microvascular endothelial cell (BMEC)-like cells on nanomembranes. The cells were differentiated by the extended endothelial cell culture method (EECM). You can find detailed information about EECM-BMEC-like cells in the previous post submitted by Hideaki Nishihara (24.10.2020).

Method

  • Coat the bottom channel and the well with 100 µl coating solution consisting of 400 μg/ml collagen IV and 100 μg/ml fibronectin. Incubate 30 min at 37°
  • Replace the coating solution at the bottom well with hECSR medium.
  • Seed 20,000 EECM-BMEC-like cells in hECSR medium into each well.
  • After ~1 hour-long incubation at 37°C, change the hECSR medium. Incubate at 37°C until the next day.
  • Change the hECSR medium at the bottom channel and the well. In case of staining for adhesion molecules, replace hECSR medium with conditioned medium from smooth muscle-like cells (SMLCs). In addition, stimulate these wells with 0.1ng/ml of TNFα + 2 IU/mL IFN‐γ for 16 hours. Incubate at 37°C for 24 hours.
  • Immunofluorescence staining protocol depends on the protein of interest:

For the staining of VE-cadherin, Claudin-5, PECAM-1, ZO-1, Occludin (Figure 1), E-Selectin and P-selectin (Figure 2); BMEC-like cells were fixed with pre-cooled methanol for 20 seconds and then rehydrated by washing with PBS. Cells were blocked in blocking buffer (5% skimmed milk containing 0.1% Triton X‐100 in PBS) for 1 hour at room temperature. Primary antibodies diluted in blocking buffer were added and incubated for 1 hour at room temperature. After three washes with PBS, cells were incubated with secondary antibodies and DAPI for 1 hour at room temperature. After washing with PBS, Mowiol (mounting medium) was added onto the cells.

For the live staining of ICAM‐1, ICAM‐2, VCAM‐1 and MCAM (Figure 2), the primary antibodies diluted in hECSR medium were added into the wells. After incubation at 37°C for 15 minutes, cells were fixed with 4% PFA in PBS for 10 minutes at room temperature. After three washes with PBS, cells were blocked in blocking buffer (5% skimmed milk containing 0.1% Triton X‐100 in PBS) for 1 hour at room temperature. Cells were incubated with secondary antibodies and DAPI for 1 hour at room temperature and then mounted by dropwise addition of Mowiol.

Results

Figure 1: Immunofluorescence staining of junctional proteins on EECM-BMEC-like cells cultivated onto nanomembrane.

 

Figure 2: Immunofluorescence staining of adhesion molecules on EECM-BMEC-like cells cultivated onto nanomembrane. *For the stainings of ICAM-1, VCAM-1, and P-selectin cells were pre-stimulated with 0.1 ng/mL TNF‐α + 2 IU/mL IFN‐γ.

Conclusion

We were able to culture EECM-BMEC-like cells in μSiM device using nanomembranes. EECM-BMEC-like cells kept their junctional protein and adhesion molecule phenotype. The next steps will be to cultivate EECM-BMEC-like cells on dual scale membranes and to check the growth of EECM-BMEC-like cells differentiated from several different donors.

Posted in Protocols (Public)