Immunofluorescent Analysis of DNA Damage in Mammospheres

The mammospheres were fixed with 4% paraformaldehyde (Sigma-Aldrich) at room temperature for 15 min. Samples were incubated with 1% Triton X-100 (Sigma-Aldrich) at room temperature for 10 min and blocked with 5% normal goat serum (Gibco). The mammospheres were attached to slides via cytospin at low speed (Shandon Cytospin 3; Shandon Scientific, Runcorn, Cheshire, UK) and stained with anti-γH2AX (#9718, 1:400 dilution, CST) and goat anti-rabbit IgG H and L labeled with Alexa Fluor 488 (Abcam) as described previously (41 (link)). Nuclei were stained with ProLong^™ Gold Antifade Mountant with DAPI (Invitrogen). Cells were imaged using a Leica THUNDER Imager 3D Cell Culture microscope, as described previously (41 (link)).

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Yamashita N., Morimoto Y., Fushimi A., Ahmad R., Bhattacharya A., Daimon T., Haratake N., Inoue Y., Ishikawa S., Yamamoto M., Hata T., Akiyoshi S., Hu Q., Liu T., Withers H., Liu S., Shapiro G.I., Yoshizumi T., Long M.D, & Kufe D. (2022). MUC1-C Dictates PBRM1-Mediated Chronic Induction of Interferon Signaling, DNA Damage Resistance, and Immunosuppression in Triple-Negative Breast Cancer. Molecular Cancer Research, 21(3), 274-289.

Publication 2022

paraformaldehyde 1% Triton X-100 normal goat serum goat anti-rabbit IgG H and L Alexa Fluor 488 ProLong™ Gold Antifade Mountant with DAPI THUNDER Imager 3D Cell Culture microscope

3d cell culture Alexa fluor 488 Anti igg Cells Dapi Dilution Goat Gold Microscope Nuclei Paraformaldehyde Rabbit Serum Triton x 100

Corresponding Organization : Roswell Park Comprehensive Cancer Center

Other organizations : Kyushu University

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Variable analysis

independent variables

Concentration of paraformaldehyde (4%)
Duration of paraformaldehyde fixation (15 min)
Concentration of Triton X-100 (1%)
Duration of Triton X-100 incubation (10 min)
Concentration of normal goat serum (5%)
Dilution of anti-γH2AX antibody (1:400)
Attachment of mammospheres to slides via cytospin

dependent variables

Staining of γH2AX in mammospheres
Staining of nuclei with DAPI

control variables

Temperature (room temperature)
Imaging using a Leica THUNDER Imager 3D Cell Culture microscope

controls

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

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