BT-549 cells were stained as described.9 (link) Briefly, cells were fixed in 4% paraformaldehyde and were incubated with 0.1% Triton X-100 at room temperature for 10 min. The samples were blocked with 3% Normal Goat Serum (Gibco), incubated with anti-MICA (PA5-35346, 1:20 dilution; Thermo Fisher Scientific) or anti-MICB (NBP2-56506, 0.9 μg/mL; Novus Biologicals, Centennial, Colorado, USA) at 4°C overnight and incubated with goat antirabbit IgG H and L labeled with Alexa Fluor 488 (Abcam). Invitrogen ProLong Gold Antifade Mountant with DAPI (Invitrogen) was used to stain the nuclei. The cells were analyzed by a Leica THUNDER Imager 3D Cell Culture microscope as described.9 (link)
Protocol detail
Immunofluorescence Staining of BT-549 Cells
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Corresponding Organization : Dana-Farber Cancer Institute
Other organizations : Nagoya University, Broad Institute, Osaka University
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Variable analysis
independent variables
- Staining treatment (anti-MICA, anti-MICB, and no primary antibody control)
- Expression of MICA and MICB proteins in BT-549 cells
- Cell type (BT-549 cells)
- Fixation and permeabilization conditions (4% paraformaldehyde, 0.1% Triton X-100)
- Blocking conditions (3% Normal Goat Serum)
- Secondary antibody (goat anti-rabbit IgG H and L labeled with Alexa Fluor 488)
- Nuclear staining (Invitrogen ProLong Gold Antifade Mountant with DAPI)
- Positive control: Cells stained with anti-MICA or anti-MICB primary antibodies
- Negative control: Cells with no primary antibody
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