25 × 103 cells per compartment of CAF1-CCK2 or CAF2-CCK2 cells were seeded into 4-compartment Cellview dishes (Greiner Bio-One) for cell proliferation or cell death analysis respectively, grown overnight and incubated with USPION@gastrin (4, 8 or 16 μgmagnetic Fe ml−1) for 24 h at 37 °C in a DMEM/F-12 medium buffered with 10 mM HEPES buffer pH 7.4 containing 0.5% FBS and 100 IU ml−1 penicillin/streptomycin. After removing the medium, the cells were rinsed twice with the incubation medium, incubated in the incubation medium with or without 10 μM CA-074 Me or 10 μM pepstatin A for 1 h, and then exposed to a magnetic field for 2 h. The effects of magnetic field treatments were investigated by labeling dead cells with Annexin V-iFluor488 (AnnV) and propidium iodide (PI) (excitation: 488 and 540 nm respectively, AAT Bioquest), 4 h after magnetic field exposure and counting them using a BD FACSCalibur™ flow cytometer.
Cellview 4 compartment dishes
Manufactured by Greiner
CELLview 4-compartment dishes are laboratory equipment designed for cell culture applications. These dishes feature four separate compartments, allowing for the simultaneous cultivation and observation of different cell types or experimental conditions within a single dish.
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5 protocols using cellview 4 compartment dishes
1
Magnetic Nanoparticle-Mediated Cell Cytotoxicity
2
Magnetic Nanoparticles Modulate Cell Proliferation
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10 × 103 cells per compartment of CAF1-CCK2 or CAF2-CCK2 cells were seeded into 4-compartment Cellview dishes (Greiner Bio-One) for cell proliferation or cell death analysis respectively, grown overnight and incubated with USPION@gastrin (4, 8 or 16 μgmagnetic Fe ml−1) for 72 h at 37 °C in a DMEM/F-12 medium buffered with 10 mM HEPES buffer pH 7.4 containing 0.5% FBS and 100 IU ml−1 penicillin/streptomycin. After removing the medium, the cells were rinsed twice with the incubation medium, and then exposed to a magnetic field for 2 h once per day. The effects of magnetic field treatments were investigated on cell proliferation by counting the cell number by using a cell counter (Beckman cell counter z2) after 24 h of RMF treatment. Cells were counted once per day for 6 days.
3
Visualization of CARD8 Protein Localization
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HEK 293T cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) were plated on CELLview 4-compartment dishes (Greiner Bio-One). HEK 293T cells were transfected with CARD8 WT and mutant UPA-CARD-mCherry constructs (0.02 μg) using lipofectamine 2000 (Thermo Fisher Scientific). Forty-eight hours after transfection, cells were fixed with 4% PFA for 10 min at RT. Cells were imaged using a spinning disk confocal Nikon microscope equipped with a Plan Apo 20×/1.3 air objective. Image analysis was performed in Fiji69 (link).
4
Immunofluorescence Localization of FLAG-tagged Proteins
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HBL1 cells cultured in fresh media were mixed in a 1:1 ratio with cytospin. Cells were spun at 800 × g for 5 minutes. Cell pellets were resuspended with cytospin and plated on CELLview 4-compartment dishes (Greiner Bio-One). Cells were left at RT overnight and were fixed with 100% cold methanol for 5 minutes at −20°C, followed by cell permeabilization with 0.1% Triton X-100 in PBS-Tween (PBST) for 10 minutes. Cells were incubated with blocking buffer containing 3% BSA for 3 hours to minimize nonspecific binding. After blocking, cells were incubated overnight at 4°C with FLAG primary antibody (Sigma-Aldrich; cat. #F1804, RRID:AB_262044). After incubation, cells were washed with PBST 3 times and incubated with AlexaFluor488-conjugated anti-mouse IgG (Abcam; cat. #ab150113, RRID:AB_2576208) for 1 hour at RT. After incubation, cells were washed with PBS and then stained with Hoechst for 10 minutes (1:500, Immunochemistry Technologies; cat. #639). Cells were imaged using a spinning disk confocal microscope with ×100 objective.
5
Immunofluorescence analysis of FLAG-tagged proteins
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