Systematic comparisons of various markers for mast cell activation in RBL-2H3 cells
6
Issued Date
2022-12-01
Resource Type
ISSN
0302766X
eISSN
14320878
Scopus ID
2-s2.0-85138347239
Pubmed ID
36125550
Journal Title
Cell and Tissue Research
Volume
390
Issue
3
Start Page
413
End Page
428
Rights Holder(s)
SCOPUS
Bibliographic Citation
Cell and Tissue Research Vol.390 No.3 (2022) , 413-428
Suggested Citation
Rujitharanawong C., Yoodee S., Sueksakit K., Peerapen P., Tuchinda P., Kulthanan K., Thongboonkerd V. Systematic comparisons of various markers for mast cell activation in RBL-2H3 cells. Cell and Tissue Research Vol.390 No.3 (2022) , 413-428. 428. doi:10.1007/s00441-022-03687-w Retrieved from: https://repository.li.mahidol.ac.th/handle/123456789/83531
Title
Systematic comparisons of various markers for mast cell activation in RBL-2H3 cells
Author's Affiliation
Other Contributor(s)
Abstract
Mast cell activation plays a key role in various allergic diseases and anaphylaxis. Several methods/techniques can be used for detection of mast cell activation. However, there was no previous systematic evaluation to compare the efficacy of each method/technique. The present study thus systematically compared various markers for mast cell activation induced by IgE cross-linking. The widely used RBL-2H3 mast cells were sensitized with anti-DNP (dinitrophenyl) IgE overnight and activated with DNP-BSA (bovine serum albumin) for up to 4 h. The untreated cells and those with anti-DNP IgE sensitization but without DNP-BSA activation served as the controls. Intracellular calcium level gradually increased to ~2-fold at 1 h, reached its peak (~5-fold) at 2 h, and returned to the basal level at 3-h post-activation. The increases in cellular tryptase level (by Western blotting) (~0.3- to 0.4-fold) and average cell size (~2.5-fold) and decrease of nucleus/cytoplasm ratio (~0.4- to 0.5-fold) were marginal at all time-points. By contrast, β-hexosaminidase release and CD63 expression (by both flow cytometry and immunofluorescence detection/localization), secreted tryptase level (by Western blotting), and tryptase expression (by immunofluorescence detection/localization) stably and obviously increased (~10-fold as compared with the untreated control and sensitized-only cells or detectable only after activation). Based on these data, the stably obvious increases (by ≥ 10-fold) in β-hexosaminidase release, CD63 expression (by both flow cytometry and immunofluorescence staining), secreted tryptase level (by Western blotting), and tryptase expression (by immunofluorescence staining) are recommended as the markers of choice for the in vitro study of mast cell activation using RBL-2H3 cells.