The following reagents were used: polyethylenimine (PEI; Sigma-Aldrich), polybrene (Santa-Cruz), PMA (Invivogen), doxycycline (Sigma-Aldrich), LPS (O111:B4; Sigma-Aldrich), nigericin (Invivogen), ATP (Sigma-Aldrich), MCC950 (Adipogen), CY-09 (Tocris), G5 (Calbiochem), CRT0066101 (Tocris), PI (Immunochemistry Technologies), Hoechst (Immunochemistry Technologies).
Hoechst
Manufactured by ImmunoChemistry Technologies
Sourced in United States
Hoechst is a fluorescent dye used for labeling and visualizing DNA in various laboratory applications. It is a membrane-permeable dye that binds to the minor groove of DNA, emitting a blue fluorescence upon excitation.
Automatically generated - may contain errors
Lab products found in correlation
Cellview 4 compartment dishes, by Greiner (2 mentions)
Flag primary antibody, by Merck Group (2 mentions)
Lps o111 b4, by Merck Group (1 mentions)
Polyethylenimine (pei), by Merck Group (1 mentions)
Adenosine triphosphate (atp), by Merck Group (1 mentions)
Doxycycline, by Merck Group (1 mentions)
Polybrene, by Santa Cruz Biotechnology (1 mentions)
Phorbol myristate acetate (pma), by InvivoGen (1 mentions)
Nigericin, by InvivoGen (1 mentions)
Crt0066101, by Bio-Techne (1 mentions)
Mcc950, by Adipogen (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Dm irb inverted fluorescence microscope, by Leica (1 mentions)
Formaldehyde, by Merck Group (1 mentions)
Sodium azide, by Merck Group (1 mentions)
Rabbit anti human irf5 antibody, by Cell Signaling Technology (1 mentions)
Cy3 labeled goat anti rabbit igg antibody, by Jackson ImmunoResearch (1 mentions)
Dfc300 fx digital color camera, by Leica (1 mentions)
Cyto id, by Enzo Life Sciences (1 mentions)
Deltavision imaging system, by GE Healthcare (1 mentions)
11 protocols using hoechst
1
Inflammation Modulation Assay Protocol
2
Immunofluorescent Detection of IRF5 Translocation
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For analysis of IRF5 translocation, DCs or macrophages were stimulated as indicated in Maxisorp plates. After 2 h stimulation, cells were washed with PBS, fixed with 3.7% formaldehyde (Sigma-Aldrich) for 15 min at room temperature, washed in PBS and stored in PBS containing 0.5% bovine serum albumin (BSA; PAA) and 0.1% sodium azide (Merck) at 4°C. Cells were permeabilized with 0.2% Triton X-100 (Sigma-Aldrich) for 5 min at room temperature and blocked for 30 min in PBS containing 0.5% BSA and 0.1% sodium azide. Cells were then stained with a rabbit-anti-human-IRF5 antibody (1:400) (Cell Signaling) or rabbit-anti-human NF-kB p65 antibody (1:100) (Cell Signaling) for 45 min at room temperature, washed with PBS and stained with a Cy3-labeled goat-anti-rabbit-IgG antibody (1:50) (Jackson ImmunoResearch). Cells were again washed with PBS and nuclei were stained using 1 μg/mL Hoechst (Immunochemistry Technologies) for 1 min at room temperature. Cells were imaged using a DM IRB inverted fluorescence microscope (Leica), combined with a DFC 300FX digital color camera (Leica).
3
Measurement of Autophagic Activity
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
Immunostaining of Fly Abdomens
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Fly abdomens were dissected in 1× PBS, fixed in 4% paraformaldehyde diluted in 0.3% 1× Phosphate-buffered saline plus Triton X-100 (PBST) for 20–30 min. Samples were washed in PBST, blocked with PBST plus Donkey or Goat Serum Albumin for 1 h, and then washed in PBST three times for 10 min each. The samples were incubated with anti-Pericardin (Developmental Studies Hybridoma Bank # EC11) at 2 μg/ml overnight at 4°C, washed in PBST three times for 10 min each, then incubated with Alexa Fluor 594 anti-mouse IgG (Jackson ImmunoResearch # 115-585-166) for 2 h at room temperature. Samples were washed in PBST three times for 10 min each. Hoechst (Immunochemistry Technologies # 639) and Alexa Fluor 488 Phalloidin (Thermo Fisher scientific # 12379) were applied for 30 min followed by washing in 1× PBS and mounted in ProLong Diamond Antifade Mountant (Invitrogen # P36961) according to manufacturer’s protocols.
6
YFV NS1 Protein Modulates SDC-1 Expression
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HPMEC (1×105 cells) were seeded on gelatin-coated coverslips and allowed to grow until full confluency was attained (approximately 3 days). On the day of the experiment, cells were treated or not with 10 μg/mL of YFV NS1 protein and incubated for 3h at 37°C. After incubation, cells were washed, fixed with 4% paraformaldehyde (PFA), and stained overnight with 2 ug/mL of the rabbit anti-SDC-1 IgG antibody (Abcam, ab188861). After washing, secondary staining was performed by adding 2 ug/mL of donkey anti-rabbit IgG conjugated to Alexa Fluor 647 (Abcam, ab150075) for 4h. Nuclei were stained using Hoechst (ImmunoChemistry Technologies). Mounted slides were imaged on a Zeiss LSM 710 Axio Observer fluorescence microscope (CRL Molecular Imaging Center, UC Berkeley). Images acquired using the Zen 2010 software (Zeiss, Jena, Germany) were processed and analyzed with ImageJ software (39 (link)). Mean fluorescence intensity (MFI) values for SDC-1 staining were obtained from individual RGB-grayscale-transformed images (n = 3).
7
Immunofluorescence analysis of FLAG-tagged proteins
8
Automated Fluorescence-Based Cell Viability Assay
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A microscopic cell-counting assay was performed using automated analysis of fluorescent microscope images. Live cells were identified by staining the cell nuclei with 1 µg/ml Hoechst (33342, ImmunoChemistry Technologies, Bloomington, MN, USA) and with 250 ng/ml propidium iodide (Sigma Aldrich, St. Louis, MO, USA), for elimination of dead cells from the analysis. At the experimental end-point, cells were pipetted in order to break up cell clusters, then spun down. Fluorescence images were taken using a Hermes microscope (IDEA Bio-Medical Ltd.) and image analysis software (WiSoft, IDEA Bio-Medical Ltd.) was used to quantify viable cell numbers.
A metabolic viability assay was performed by adding 20 µl CellTiter-Blue (Promega Corporation, Madison, WI, USA) per 100 µl culture medium for 3 h. Results were quantified using a fluorescence plate reader (excitation 560 nm; emission 590 nm), and a linear equation of a calibration column.
A metabolic viability assay was performed by adding 20 µl CellTiter-Blue (Promega Corporation, Madison, WI, USA) per 100 µl culture medium for 3 h. Results were quantified using a fluorescence plate reader (excitation 560 nm; emission 590 nm), and a linear equation of a calibration column.
9
Visualizing NLRP3 Variant Localization
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To follow the localization of the NLRP3 variants, stable Nlrp3−/− cell lines encoding NLRP3-YFP (full-length mouse NLRP), (1–665)-YFP, or (1–686)-YFP cells were seeded into a μ-slide (Ibidi) (3 × 105 per well). Cells were non-treated or treated with LPS (100 ng/ml) and doxycycline (1 μg/ml) overnight. In the morning, the cells were washed and incubated with Hoechst (1 μg/ml; Immunochemistry Technologies) for 5 min at 37 °C, and washed several times before nigericin was added (5 μM). After 1 h, the cells were observed under a Leica TCS SP5 laser scanning microscope mounted on a Leica DMI 6000 CS inverted microscope (Leica Microsystems, Germany) with an HCX plan apo 63 × (NA 1.4) oil immersion objective used for imaging. A 405-nm laser line of a 20-mW diode laser was used for Hoechst excitation, and emitted light was detected between 450 and 510 nm. A 514-nm laser line of an 100-mW argon laser with 25% laser power was used to detect YFP, where emitted light was detected between 530 and 600 nm. To acquire and process the images, Leica LAS AF software was used.
10
Assessing Cell Permeabilization and Pyroptosis
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To assess cell permeabilization, cells were cultured in media without phenol red. PI (1.25 μg/ml, Immunochemistry technologies), YOPROTM-1 Iodide (1 μM, Invitrogen) and Hoechst (0.2 μg/ml, Immunochemistry technologies) were added 1 h before time-lapse imaging using CQ1 high content screening microscope (Yokogawa). Twi images/well were taken every 10 min using ×10 objectives. Images were quantified using the CQ1 software (Yokogawa). Pyroptosis was assessed using Cytotoxicity Detection KitPLUS (LDH) (Roche) according to the manufacturer’s instructions. Values are available in the Source Data file.
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