2.4. Vascular permeability and Evans blue dye assay

2.4. Vascular permeability and Evans blue dye assay¹

AIM:  We used EB staining to compare a plausible impact of alterations in vascular permeability in the homeostasis of Bmi1+ cardiac progenitor cells. Histamine treatment alters the integrity of vessels^1,2.

Protocol

Mice

· We used C57BL/6 mice (wt and mutant animals); 8-12 weeks old (20 g mean body weight).  n= 5-7 mice /group

note: the ideal situation is to select animals with the same body weight to use the same injection volume per animal

Materials & equipment

· Standard molecular biology material and syringes (10 mL)

· 96-well plate spectrophotometer (read at 620 and 720 nm)

Reagents

· Histamine (Sigma, H7125) in phosphate-buffered saline (PBS); 

Stock solution: 25 mg/ml. Filtered (0.4 mM), aliquoted and keep frozen (-20ºC). 

· Evans Blue (EB; Sigma, E2129) dye solution; 0.5% w/v EB in PBS. Filtered (0.4 mM) and used freshly prepared. 

· Sterile PBS

· Formamide (Millipore, 109684).

· Prepare a standard curve of EB in formamide.

Methods

· Histamine administration

√ Mice with injected (via i.v.) with histamine (2.5 mg /mouse; 100 ml Stock solution) or PBS (negative control), 4 h before EB staining.

· Evans Blue administration and evaluation

√ Mice are injected (via i.v.) with the Evans Blue (EB) dye (100 μl).

√ 30 min later mice were euthanized and hearts were perfused with 10 ml of PBS (using a syringe and at low rate) through the left ventricle to remove the excess EB from vessels¹. Hearts, cut in half, were introduced in Eppendorf tubes (2mL) and desiccated (24 h, 60 °C; in a conventional stove). 

√ EB dye was then eluted (1-1.5 ml per heart) in formamide (24 h, 65 °C; moderate sacking).

√ To quantitate the amount of EB in the supernatant, samples (in triplicates) were placed in a 96-well plate and read with a Multiskan GO Spectrophotometer (Thermo Scientific) at 620 and 720 nm, correcting for contaminating heme pigments: 

A620 (EB)=A620-(1.426 Å~ A720+ 0.030)¹. 

The EB concentration was calculated by interpolation in the standard curve. 

References

1. Herrero D, Cañón S, Albericio G, Carmona RM, Aguilar S, Mañes S, Bernad A. Age-related oxidative stress confines damage-responsive Bmi1⁺ cells to perivascular regions in the murine adult heart. Redox Biol  (2019):101156. https://doi:10.1016/j.redox.2019.101156.

2.  K. Ashina, Y. Tsubosaka, T. Nakamura, K. Omori, K. Kobayashi, M. Hori, T. Murata, Histamine induces vascular hyperpermeability by increasing blood flow and endothelial barrier disruption in vivo, PLoS One 10 (7) (2015)  e0132367, https://doi.org/10.1371/journal.pone.013236.

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