Cells were transfected as experiments designed. After transfection 48 h, cells were stained with EdU solution (Abcam, iFluor 647) for 4 h. Then cells were incubated with reaction mix to label EdU fluorescently. The stained cells were analyzed by the fluorescence microscope.
Ifluor 647
Manufactured by Abcam
Sourced in United Kingdom, United States
IFluor 647 is a fluorescent dye that can be used for labeling and detection applications in biological research. It has an excitation maximum at 650 nm and an emission maximum at 665 nm, making it suitable for detection in the far-red/near-infrared region of the spectrum.
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Lab products found in correlation
Fluorescence microscopy, by Olympus (1 mentions)
Fv1000 ix81, by Olympus (1 mentions)
Cytoflex lx, by Beckman Coulter (1 mentions)
96 well plate, by Corning (1 mentions)
Mouse anti α tubulin dm1α, by Merck Group (1 mentions)
Mouse anti tau1, by Merck Group (1 mentions)
Zenon rabbit igg labeling kit, by Thermo Fisher Scientific (1 mentions)
Rabbit anti myosin iib, by Fortrea (1 mentions)
Eclipse c1, by Nikon (1 mentions)
Ab176759, by Abcam (1 mentions)
A11070, by Thermo Fisher Scientific (1 mentions)
Imagexpress pico high content imaging system, by Molecular Devices (1 mentions)
Mtt assay kit, by Dojindo Laboratories (1 mentions)
10 protocols using ifluor 647
1
Quantifying Cell Proliferation by EdU Assay
2
Cell Proliferation Measurement with EdU Luminescence
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An EdU luminescence kit (iFluor 647, Abcam Inc., Cambridge, UK) was applied for cell proliferation measurement in strict accordance with the instructions. The stably transfected cells (5 × 103 cells/well) were cultured in a 96-well petri dish for 24 h. Then, the cells in each well were cultured with 100 μL culture medium containing 20 μm EdU at 37°C for 2 h. The number of EdU positive cells was observed and calculated by a fluorescence microscopy (Olympus Optical Co., Ltd., Tokyo, Japan) [16 (link)].
3
BT549 Cell Proliferation Assay
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We investigated the proliferation of BT549 cells treated with THZ1, anti-PD-L1 antibody, and their combination using a Cell-Light EdU Cell Proliferation Kit (Abcam), according to the manufacturer's instructions. Briefly, the BT549 cells were seeded into the wells of a 24-well plate at a density of 2 × 105 per well. The cells of THZ1 (MedChem Express) and the combination groups were treated with 50 nmol/L THZ1 for 24 h. Anti-PD-L1 antibody (clone SP263, rabbit, Ventana) was added to fresh media at a final concentration of 10 μg/mL. After 1 h, the freshly isolated CD8+ T cells were added to the media, and the ratio of CD8+ T cells and BT549 cells was 5:1, and 24 h later, the medium was replaced with 100 μL of 10 μmol/L EdU medium in each well. The cells were then incubated for 2 h and fixed with 4% paraformaldehyde. They were permeated with 0.5% Triton X-100 for 10 min and were stained with iFluor 647 (Abcam) for 30 min. The cells were subsequently counterstained with 4′,6-diamidino-2-phenylindole (DAPI) and imaged via fluorescence microscopy (Olympus, Tokyo, Japan).
4
Quantifying Cell Proliferation with EdU
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An EdU proliferation kit (iFluor647, Abcam) was used to detect cell proliferation. Following the protocols described earlier (Liu et al., 2019 (link)), we seeded vector-GFP- and circ-ATXN2–GFP-expressing cells (1 × 104) onto 96-well plates (Corning) for 24 h. The cells were treated with EdU solution (50 µM) for 4 h at 37°C. Thereafter, the cells were fixed with 4% paraformaldehyde. Cells were then permeabilized with PBS supplemented with 0.5% Triton X-100 for 10 min and stained with Hoechst 33,342 for 30 min in the dark. Finally, the cells were viewed under a confocal microscope (IX81-FV1000, Olympus, Japan).
For flow cytometry, vector-GFP- and circ-ATXN2–GFP-expressing cells (5 × 105) were seeded into 6-well plates for 24 h. Following the above methods, a flow cytometer (Cytoflex LX, Beckman Coulter, United States) was employed to detect the percentage of EdU-positive cells.
For flow cytometry, vector-GFP- and circ-ATXN2–GFP-expressing cells (5 × 105) were seeded into 6-well plates for 24 h. Following the above methods, a flow cytometer (Cytoflex LX, Beckman Coulter, United States) was employed to detect the percentage of EdU-positive cells.
5
Immunostaining of Neuronal Cytoskeleton Proteins
6
Immunohistochemistry for F-Actin Visualization
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The sections were permeabilized three times with 0.2% Triton X‐100 and incubated in sequence with primary antibodies and appropriate conjugated secondary antibodies. All antibodies were same as used in WB assay. In addition, F‐Actin was stained with iFluor 647 (ab176759, 1:1000, Abcam) in brain paraffin sections and HBMECs. The images were viewed on a confocal microscope (Nikon, Nikon Eclipse C1).
7
Evaluating Cancer Cell Proliferation
8
Endothelial Cell Stress Response
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Cells were cultured on coverslips in 24-well plates. Upon reaching confluence, cells were challenged with hemoglobin, ferryl hemoglobin, and Peptides 1–5 for 7 h. Cells were fixed with 3.7% formaldehyde for 15 min. F-Actin was stained with iFluor 647 (ab176759, Abcam), Hoechst (33258) was used to stain nuclei. To show NF-kβ nuclear translocation, cells were challenged with Peptides 1–5, hemoglobin, and ferryl hemoglobin for 1.5 h. After treatment, the cells were fixed with 3.7% formaldehyde for 15 min. After fixation, the cells were blocked with 5% goat serum for 1 h at room temperature. Rabbit monoclonal antihuman NF-kβ (701079, Thermo Scientific, diluted 1:25) was used as a primary antibody to show NF-kβ nuclear translocalization in endothelial cells. AF488-labeled goat anti-rabbit secondary antibody (A11070, Thermo Scientific, diluted 1:500) was incubated with the cells for 1 h in the dark at room temperature. F-Actin was stained with iFluor 647 (ab176759, Abcam). Hoechst (33258) was used to stain nuclei.
9
HT29 Cell Proliferation Assay
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Proliferation rate of HT29 cells was determined using a 5-ethynyl-2’-deoxyuridine (EdU) Staining Proliferation kit (iFluor 647) (Abcam) (Benoit et al., 2021 (link)). Briefly, 5,000 cells per well were plated in 96-well plates. 24 hours post-seeding, cells were treated with CWP232228 or YB-0158 for 48 hours vs. DMSO control. Two hours before the end of the drug incubation period, a pulse of EdU was added to all cultures, according to manufacturer’s protocol. Then, cells were formalin-fixed (2% v/v) and EdU-positive cells were fluorescently labeled following a click chemistry reaction. Nuclei were stained with Hoechst 33342. Image acquisition and EdU-positive cell scoring was done using an ImageXpress Pico High-Content imaging system (Molecular Devices), as described above.
10
Assessing Cell Viability and Proliferation
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Well growing H1299 and A549 cells were harvested. The viability of cells was determined using a 3-(4, 5dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay kit (Dojindo Laboratories, Kumamoto, Japan), while cell proliferation was determined using a 5-ethynyl-2'-deoxyuridine (EdU) labeling assay kit (iFluor 647, Abcam Co., Ltd., NY, USA). All procedures were conducted in strict accordance with the manufacturer's instructions. Transwell assays was performed to measure invasion and migration of cells as previously described [17] .
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