Protocol for vital dye staining of corneal endothelial cells

Sunju Park, Alan G. Fong, Hyung Cho, Cheng Zhang, David C. Gritz, Gibran Mian, Alexandra A. Herzlich, Patrick Gore, Ashley Morganti, Roy S. Chuck

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

Purpose: To describe a step-by-step methodology to establish a reproducible staining protocol for the evaluation of human corneal endothelial cells. Methods: Four procedures were performed to determine the best protocol. (1) To determine the optimal trypan blue staining method, goat corneas were stained with 4 dilutions of trypan blue (0.4%, 0.2%, 0.1%, and 0.05%) and 1% alizarin red. (2) To determine the optimal alizarin red staining method, goat corneas were stained with 2 dilutions of alizarin red (1% and 0.5%) and 0.2% trypan blue. (3) To ensure that trypan blue truly stains damaged cells, goat corneas were exposed to either 3% hydrogen peroxide or to balanced salt solution, and then stained with 0.2% trypan blue and 0.5% alizarin red. (4) Finally, fresh human corneal buttons were examined; 1 group was stained with 0.2% trypan blue and another group with 0.4% trypan blue. Results: For the 4 procedures performed, the results are as follows: (1) trypan blue staining was not observed in any of the normal corneal samples; (2) 0.5% alizarin red demonstrated sharper cell borders than 1% alizarin red; (3) positive trypan blue staining was observed in the hydrogen peroxide exposed tissue in damaged areas; (4) 0.4% trypan blue showed more distinct positive staining than 0.2% trypan blue. Conclusions: We were able to determine the optimal vital dye staining conditions for human corneal endothelial cells using 0.4% trypan blue and 0.5% alizarin red.

Original languageEnglish (US)
Pages (from-to)1476-1479
Number of pages4
JournalCornea
Volume31
Issue number12
DOIs
StatePublished - Dec 2012

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Trypan Blue
Coloring Agents
Endothelial Cells
Staining and Labeling
Goats
Cornea
Hydrogen Peroxide
alizarin
Salts

Keywords

  • alizarin red
  • corneal transplant
  • trypan blue

ASJC Scopus subject areas

  • Ophthalmology

Cite this

Protocol for vital dye staining of corneal endothelial cells. / Park, Sunju; Fong, Alan G.; Cho, Hyung; Zhang, Cheng; Gritz, David C.; Mian, Gibran; Herzlich, Alexandra A.; Gore, Patrick; Morganti, Ashley; Chuck, Roy S.

In: Cornea, Vol. 31, No. 12, 12.2012, p. 1476-1479.

Research output: Contribution to journalArticle

Park, S, Fong, AG, Cho, H, Zhang, C, Gritz, DC, Mian, G, Herzlich, AA, Gore, P, Morganti, A & Chuck, RS 2012, 'Protocol for vital dye staining of corneal endothelial cells', Cornea, vol. 31, no. 12, pp. 1476-1479. https://doi.org/10.1097/ICO.0b013e31824d0dda
Park, Sunju ; Fong, Alan G. ; Cho, Hyung ; Zhang, Cheng ; Gritz, David C. ; Mian, Gibran ; Herzlich, Alexandra A. ; Gore, Patrick ; Morganti, Ashley ; Chuck, Roy S. / Protocol for vital dye staining of corneal endothelial cells. In: Cornea. 2012 ; Vol. 31, No. 12. pp. 1476-1479.
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AU - Cho, Hyung

AU - Zhang, Cheng

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AU - Mian, Gibran

AU - Herzlich, Alexandra A.

AU - Gore, Patrick

AU - Morganti, Ashley

AU - Chuck, Roy S.

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N2 - Purpose: To describe a step-by-step methodology to establish a reproducible staining protocol for the evaluation of human corneal endothelial cells. Methods: Four procedures were performed to determine the best protocol. (1) To determine the optimal trypan blue staining method, goat corneas were stained with 4 dilutions of trypan blue (0.4%, 0.2%, 0.1%, and 0.05%) and 1% alizarin red. (2) To determine the optimal alizarin red staining method, goat corneas were stained with 2 dilutions of alizarin red (1% and 0.5%) and 0.2% trypan blue. (3) To ensure that trypan blue truly stains damaged cells, goat corneas were exposed to either 3% hydrogen peroxide or to balanced salt solution, and then stained with 0.2% trypan blue and 0.5% alizarin red. (4) Finally, fresh human corneal buttons were examined; 1 group was stained with 0.2% trypan blue and another group with 0.4% trypan blue. Results: For the 4 procedures performed, the results are as follows: (1) trypan blue staining was not observed in any of the normal corneal samples; (2) 0.5% alizarin red demonstrated sharper cell borders than 1% alizarin red; (3) positive trypan blue staining was observed in the hydrogen peroxide exposed tissue in damaged areas; (4) 0.4% trypan blue showed more distinct positive staining than 0.2% trypan blue. Conclusions: We were able to determine the optimal vital dye staining conditions for human corneal endothelial cells using 0.4% trypan blue and 0.5% alizarin red.

AB - Purpose: To describe a step-by-step methodology to establish a reproducible staining protocol for the evaluation of human corneal endothelial cells. Methods: Four procedures were performed to determine the best protocol. (1) To determine the optimal trypan blue staining method, goat corneas were stained with 4 dilutions of trypan blue (0.4%, 0.2%, 0.1%, and 0.05%) and 1% alizarin red. (2) To determine the optimal alizarin red staining method, goat corneas were stained with 2 dilutions of alizarin red (1% and 0.5%) and 0.2% trypan blue. (3) To ensure that trypan blue truly stains damaged cells, goat corneas were exposed to either 3% hydrogen peroxide or to balanced salt solution, and then stained with 0.2% trypan blue and 0.5% alizarin red. (4) Finally, fresh human corneal buttons were examined; 1 group was stained with 0.2% trypan blue and another group with 0.4% trypan blue. Results: For the 4 procedures performed, the results are as follows: (1) trypan blue staining was not observed in any of the normal corneal samples; (2) 0.5% alizarin red demonstrated sharper cell borders than 1% alizarin red; (3) positive trypan blue staining was observed in the hydrogen peroxide exposed tissue in damaged areas; (4) 0.4% trypan blue showed more distinct positive staining than 0.2% trypan blue. Conclusions: We were able to determine the optimal vital dye staining conditions for human corneal endothelial cells using 0.4% trypan blue and 0.5% alizarin red.

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