Immunofluorescent Analysis of Mammospheres

Mammospheres were fixed with 4% paraformaldehyde (Sigma-Aldrich) at room temperature for 15 minutes. Samples were incubated with 1% Triton X-100 (Sigma-Aldrich) at room temperature for 10 minutes and blocked with 5% normal goat serum (Gibco). The mammospheres were attached to slides via cytospin at low speed (Shandon Cytospin 3; Shandon Scientific) 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 (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 (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 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

Fixation with 4% paraformaldehyde (Sigma-Aldrich) at room temperature for 15 minutes
Incubation with 1% Triton X-100 (Sigma-Aldrich) at room temperature for 10 minutes

dependent variables

γH2AX expression (stained with anti-γH2AX (#9718, 1:400 dilution, CST) and goat anti-rabbit IgG H and L labeled with Alexa Fluor 488 (Abcam))
Nuclei staining with ProLong Gold Antifade Mountant with DAPI (Invitrogen)

control variables

Blocking with 5% normal goat serum (Gibco)
Attachment of mammospheres to slides via cytospin at low speed (Shandon Cytospin 3; Shandon Scientific)

controls

Not specified
Not specified

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