Immunofluorescence Quantification of SMN Gems

For immunofluorescence, cells were seeded onto gelatinized glass coverslips at a density of 4000 cells/cm² and treated with compounds as described above. Immunostaining of fibroblast cells was accomplished as described previously [19 (link);35 (link)] using the MANSMA2 mouse anti-SMN mAb (1:200; Developmental Studies Hybridoma Bank, Iowa City, IA [36 (link)]). SMN immunostaining within the nuclei of treated fibroblasts was visualized using a DMRXA2 epifluorescence microscope (Leica Microsystems) with an ORCA-ER cooled camera (Hamamatsu, Hamamatsu City, Japan) and Volocity 6.1.1 software (Perkin-Elmer). Gems were counted 10 randomly selected nuclei in a field of view; this process was repeated for a total of 10 randomly selected, non-overlapping fields of view. The following parameters were measured: the number of gems, the number of cells with gems and the number of cells with more than 1 gem.

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Gentillon C., Connell A.J., Kirk R.W, & Butchbach M.E. (2017). The effects of C5-substituted 2,4-diaminoquinazolines on selected transcript expression in spinal muscular atrophy cells. PLoS ONE, 12(6), e0180657.

Publication 2017

DMRXA2 epifluorescence microscope ORCA-ER cooled camera Volocity 6.1.1 software

Cells Fibroblasts Gems Hybridoma Immunofluorescence Microscope Mouse Nuclei Orca

Corresponding Organization : Thomas Jefferson University

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Variable analysis

independent variables

Compounds (not specified)

dependent variables

Number of gems
Number of cells with gems
Number of cells with more than 1 gem

control variables

Cell density (4000 cells/cm^2)
Cell type (fibroblast cells)
Substrate (gelatinized glass coverslips)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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