PDL1 t-haNK, MDA-MB-231 and PD-L1 null MDA-MB-231 cells were stained as described.9 (link) The following were used as primary antibodies: NKG2d (Abcam) and perforin (Abcam), PD-L1 (Abcam). The following were used as secondary antibodies: goat anti-rabbit-AlexFLuor647 (Thermo Fisher), goat anti-mouse-AlexFLuor488 (Thermo Fisher), and goat-anti-rabbit AlexaFluor488 (Thermo Fisher). The samples were mounted using ProLong gold antifade mountant with 4′,6-diamidino-2-phenylindole (DAPI) (Thermo Fisher). The samples were imaged on a Leica DMI4000B microscope (Leica Microsystems).
Protocol detail
Immunofluorescence Staining of PD-L1 and NK Markers
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Corresponding Organization : Center for Cancer Research
Other organizations : National Institute on Deafness and Other Communication Disorders, ImmunityBio (United States)
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Variable analysis
independent variables
- PD-L1 expression (PD-L1 t-haNK vs. PD-L1 null MDA-MB-231 cells)
- NKG2d expression
- Perforin expression
- Antibodies used for staining (NKG2d, perforin, PD-L1)
- Secondary antibodies (goat anti-rabbit-AlexFLuor647, goat anti-mouse-AlexFLuor488, goat-anti-rabbit AlexaFluor488)
- Mounting medium (ProLong gold antifade mountant with DAPI)
- Imaging platform (Leica DMI4000B microscope)
- Positive control: PD-L1 t-haNK cells
- Negative control: PD-L1 null MDA-MB-231 cells
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