Cryo 2

Manufactured by Adipogen

Sourced in United States

Cryo-2 is a laboratory equipment designed for cryogenic storage and preservation of biological samples. It utilizes liquid nitrogen to maintain extremely low temperatures, ensuring the integrity and viability of stored materials.

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Lab products found in correlation

Sc 22514 r, by Santa Cruz Biotechnology (4 mentions) Al177, by Adipogen (4 mentions) Anti il 1β, by R&D Systems (3 mentions) Sc 514, by Santa Cruz Biotechnology (2 mentions) Enhanced chemiluminescence reagent, by Thermo Fisher Scientific (2 mentions) Protease inhibitor cocktail, by Roche (2 mentions) Ab179515, by Abcam (2 mentions) Anti β actin, by Cell Signaling Technology (2 mentions) Anti β actin, by Abcam (2 mentions) Af 401, by R&D Systems (2 mentions) Epr16883, by Abcam (2 mentions) Epr19828, by Abcam (2 mentions) Casper 1, by Adipogen (2 mentions) Lsm 510, by Zeiss (2 mentions) β actin, by Merck Group (2 mentions) Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (2 mentions) Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions) Fluorescence conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions) Precision plus protein dual color standard marker, by Bio-Rad (1 mentions)

Close spelling variants of this product

Anti-NLRP3 (clone Cryo-2; (4 citations) NLRP3 (Cryo-2) (4 citations) Anti-NLRP3 (Cryo-2, (3 citations) Mouse anti-human NLRP3 (Cryo-2, (2 citations) Cryo-2 AG-20B-0014 (2 citations)

26 protocols using cryo 2

Visualizing NLRP3 and ASC Specks in CTL-Stimulated DCs

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Cells were cultured on glass in complete medium. For staining ASC speck, LPS-primed and ova-pulsed BMDCs on coverslips were washed twice with PBS, incubated with CTLs that were pre-labelled with Alexa Fluor 647-CD8α (eBioscience) and fixed for 15 min at room temperature with 4% formaldehyde in PBS, and then were washed three times with PBS. After permeabilization with 0.1% Triton X-100 in PBS and blocking with 5% BSA in PBS for 1 h, cells were incubated with anti-NLRP3 (Cryo-2; AdipoGen) and anti-ASC (sc-22514-R; Santa Cruz Biotechnology, Inc.) primary antibodies (in 5% BSA) for 1 h at room temperature. Cells were washed three times with PBS and then were incubated for 1 h at room temperature with the appropriate fluorescence-conjugated secondary antibody (Invitrogen). The samples were washed three times in PBS, and then incubated with DAPI for 5 min. Samples
were observed with a Zeiss LSM510.

Yao Y., Chen S., Cao M., Fan X., Yang T., Huang Y., Song X., Li Y., Ye L., Shen N., Shi Y., Li X., Wang F, & Qian Y. (2017). Antigen-specific CD8+ T cell feedback activates NLRP3 inflammasome in antigen-presenting cells through perforin. Nature Communications, 8, 15402.

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Immunoblotting Analysis of Circadian Clock and Inflammatory Proteins

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Protein lysates were prepared using CelLytic MT Cell Lysis Reagent (Sigma) as described previously^{14 (link)}, then subjected to SDS-PAGE, with the Precision Plus Protein Dual Color Standard marker (Bio-Rad) and transferred to PVDF membrane (Bio-Rad). Primary antibodies used were REV-ERBα (mouse monoclonal, clone 4F6; 1:1000; Abgent), or REV-ERBβ (mouse monoclonal, clone D-8; 1:1000; Santa Cruz Biotechnology), or NLRP3 (mouse monoclonal, clone Cryo-2; 1:1000; Adipogen), or IL-1β (rabbit polyclonal, GTX74034; 1:1000; GeneTex), and incubated overnight at 4 °C. β-actin (mouse monoclonal, clone C4; 1:10,000; Millipore) was used as a loading control. Membranes were washed with tris-buffered saline with 0.1% Tween-20 (TBS-T), and incubated for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibodies (anti-mouse, 7076S, 1:5000; anti-rabbit, 7074S, 1:5000; Cell Signaling) in 3% skim milk in TBS-T. Proteins were visualized using Clarity Western ECL substrate (Bio-Rad), according to the manufacturer’s specifications. Densitometry quantification was performed using Image J 1.46 (NIH). All original blots are provided in Supplementary Fig. 5.

Reitz C.J., Alibhai F.J., Khatua T.N., Rasouli M., Bridle B.W., Burris T.P, & Martino T.A. (2019). SR9009 administered for one day after myocardial ischemia-reperfusion prevents heart failure in mice by targeting the cardiac inflammasome. Communications Biology, 2, 353.

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Immunofluorescence Imaging of NLRP3, Caspase-1, and ASC

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J774A.1 cells were stimulated as described above. After washing with PBS, the cells were fixed and permeated with 70–30 acetone–methanol as previously described (45 (link)). Briefly, cells were placed in a humidified slide chamber blocked for 1 h in 10% normal donkey serum (Jackson Immunologicals). After removal of the donkey serum, the primary antibodies to NLRP3 (Cryo-2; AdipoGen), caspase-1 (sc-514; Santa Cruz Biotechnology), and ASC (D2W8U; Cell Signaling) or isomolar, species-specific anti-IgG (R&D Systems, Inc.) were added and incubated overnight at 4 °C. The slides were then washed three times with PBS, and cells were incubated for 1 h at room temperature with donkey anti-mouse–Alexa555 (Life Technologies) or donkey anti-rabbit–Alexa488 (Life Technologies) conjugated secondary antibodies. Nuclei were stained with DAPI (Life Technologies). FRET images were acquired using a Marianas Imaging Station (Intelligent Imaging Innovations) using a Zeiss 639 Plan-Apochromat objective (1.4 N.A.), a Sutter Xenon light source, and a Cooke SensiCam (The Cooke Corporation) as previously described (45 (link)). The FRET was calculated as FRET = Transfer − (Fd/D) − (Fa/Aa) as reported (46 (link)).

Marchetti C., Swartzwelter B., Gamboni F., Neff C.P., Richter K., Azam T., Carta S., Tengesdal I., Nemkov T., D’Alessandro A., Henry C., Jones G.S., Goodrich S.A., St. Laurent J.P., Jones T.M., Scribner C.L., Barrow R.B., Altman R.D., Skouras D.B., Gattorno M., Grau V., Janciauskiene S., Rubartelli A., Joosten L.A, & Dinarello C.A. (2018). OLT1177, a β-sulfonyl nitrile compound, safe in humans, inhibits the NLRP3 inflammasome and reverses the metabolic cost of inflammation. Proceedings of the National Academy of Sciences of the United States of America, 115(7), E1530-E1539.

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NLRP3 Protein Expression Analysis

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To assess NLRP3 protein levels, all U937 cells were differentiated with PMA (50 ng/ml, 16 h) and treated with LPS (40 ng/ml, 3 h) or doxycycline (0.5–2 μg/ml, 3 h). Cells were directly lysed in sample buffer 2X. Samples were analyzed by SDS-PAGE and transferred to polyvinylidene difluoride membranes. The following antibodies were used: anti-NLRP3 (Cryo2; Adipogen), anti-IL-1β (D3U3E; Cell Signaling), anti-IL-18 (D2F3B; Cell Signaling), anti-LaminB (B-10; Santa Cruz Biotechnology), anti-actin (C4; Sigma-Aldrich), HRP-anti-Mouse IgG (H+L) (Promega).

Cosson C., Riou R., Patoli D., Niu T., Rey A., Groslambert M., De Rosny C., Chatre E., Allatif O., Henry T., Venet F., Milhavet F., Boursier G., Belot A., Jamilloux Y., Merlin E., Duquesne A., Grateau G., Savey L., Jacques Maria A.T., Pagnier A., Poutrel S., Lambotte O., Mallebranche C., Ardois S., Richer O., Lemelle I., Rieux-Laucat F., Bader-Meunier B., Amoura Z., Melki I., Cuisset L., Touitou I., Geyer M., Georgin-Lavialle S, & Py B.F. (2024). Functional diversity of NLRP3 gain-of-function mutants associated with CAPS autoinflammation. The Journal of Experimental Medicine, 221(5), e20231200.

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Protein Expression and Quantification

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Cells were lysed with RIPA lysis buffer, supplemented with protease and phosphatase inhibitors (both from Roche Diagnostics) and fractionated using the NE-PER Nuclear and Cytoplasmic extraction reagents (Thermo Scientific). Supernatants were filtered and concentrated by trichloroacetic acid precipitation, followed by aceton desiccation. Proteins were run on SDS-PAGE (4–12% Bis-Tris gels, Invitrogen), blotted onto PVDF membranes (GE Healthcare) blocked with 5% bovine serum albumin (BSA) or non-fat dry milk (NFDM), incubated with primary antibody: rabbit anti-IL-1 beta (1:2,500 in 5% NFDM, ab9722, Abcam), rabbit anti-ASC (1:200 in 5% BSA, sc-22514-R, Santa Cruz), mouse anti-NLRP3/NALP3 (1:1,000 in 5% milk, Cryo-2, Adipogen) or rabbit anti-MMP7 (1:200 in 5% BSA, ab4044, Abcam), washed with PBS tween 0.1% and incubated with secondary antibodies in 5% NFDM (goat anti rabbit-HRP or goat anti-mouse-HRP, Cell Signaling). Bands were imaged using ECL Plus detection reagent (GE Health Care) and quantified using ImageJ. GAPDH (1:1,000, sc-25778, Santa Cruz) was used as loading control.

Ambite I., Puthia M., Nagy K., Cafaro C., Nadeem A., Butler D.S., Rydström G., Filenko N.A., Wullt B., Miethke T, & Svanborg C. (2016). Molecular Basis of Acute Cystitis Reveals Susceptibility Genes and Immunotherapeutic Targets. PLoS Pathogens, 12(10), e1005848.

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Antibody Electroporation in Human MDMs

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Human MDMs were electroporated with PBS, mouse anti-GFP (9F9.F9; Abcam) or mouse anti-NLRP3 (Cryo-2; Adipogen) IgG antibodies. All antibodies used for electroporation were passed through Amicon Ultra-0.5 100 kDa centrifugal filter devices (Millipore) to remove traces of azide and replace buffer with PBS. All antibodies were diluted to 0.6 mg/mL in PBS prior to electroporation. Antibody electroporation was performed using the Neon Transfection System (Thermo Fisher Scientific). MDMs were washed with PBS and resuspended in Buffer R (Thermo Fisher Scientific) at a concentration of 1.4 × 10⁸ cells/mL. For each electroporation reaction 1.4 × 10⁶ cells (10 μl) were mixed with 2 μl of antibody or PBS. The mixture was taken up into a 10 μl Neon® Pipette Tip (Thermo Fisher Scientific) and electroporated using the following settings: 1400V, 20 ms, 2 pulses. Electroporated cells were transferred to growth medium without antibiotics.

Castro-Dopico T., Dennison T.W., Ferdinand J.R., Mathews R.J., Fleming A., Clift D., Stewart B.J., Jing C., Strongili K., Labzin L.I., Monk E.J., Saeb-Parsy K., Bryant C.E., Clare S., Parkes M, & Clatworthy M.R. (2019). Anti-commensal IgG Drives Intestinal Inflammation and Type 17 Immunity in Ulcerative Colitis. Immunity, 50(4), 1099-1114.e10.

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NLRP3 Ubiquitination Detection Protocol

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NLRP3 ubiquitination was detected in control and TGF-β1 stimulated cells lysed in 1% NP-40 buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 5 mM EDTA, 1% NP-40, 1 mM PMSF, 1 mM Na₃VO₄, Roche protease inhibitors) or RIPA buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 1 mM EDTA, 0.05% sodium deoxycholate, 1% Triton X-100, 0.1% SDS, 1 mM PMSF, Roche protease inhibitors). NLRP3 was immunoprecipitated overnight at 4 °C using 0.01 μg/μl of anti-NLRP3 antibody (Cryo-2, Adipogen) and 10 μl of packed protein G sepharose beads (GE Healthcare) per 100 μl of lysate. Immunoprecipitated complexes were then washed extensively in lysis buffer and proteins were separated by SDS-PAGE and analyzed by immunoblotting.

Chun J., Chung H., Wang X., Barry R., Taheri Z.M., Platnich J.M., Ahmed S.B., Trpkov K., Hemmelgarn B., Benediktsson H., James M.T, & Muruve D.A. (2016). NLRP3 Localizes to the Tubular Epithelium in Human Kidney and Correlates With Outcome in IgA Nephropathy. Scientific Reports, 6, 24667.

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NLRP3/ASC Interaction and Ubiquitination Detection

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To detect NLRP3/ASC interaction, cells were lysed with IP buffer (1% NP-40, 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.5% sodium deoxycholate, and complete protease and phosphatase inhibitor cocktail) and sonicated for 5 sec at 20 amp. The lysates were precleared and incubated with ASC antibody (sc-22514-R, Santa Cruz)^{46 (link)} overnight at 4 °C^{46 (link)}.
To detect NLRP3 ubiquitination, BMDMs were lysed with TTNE. The lysates were precleared and incubated with NLRP3 antibody overnight at 4 °C. the beads were rinsed with lysis buffer and eluted (0.1 M Tris-HCl, pH 6.8, 0.4% sodium dodecyl sulfate, 12% glycerol, 0.286 M β-mercaptoethanol, and 0.32% bromophenol blue)^{35 (link)}. The lysates were precleared and incubated with NLRP3 antibody (Cryo-2, Adipogen)^{35 (link)} overnight at 4 °C.

Shim D.W., Shin W.Y., Yu S.H., Kim B.H., Ye S.K., Koppula S., Won H.S., Kang T.B, & Lee K.H. (2017). BOT-4-one attenuates NLRP3 inflammasome activation: NLRP3 alkylation leading to the regulation of its ATPase activity and ubiquitination. Scientific Reports, 7, 15020.

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Western Blot Analysis of Inflammatory Proteins

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PBMCs, monocytes or macrophages were lysed in (5×) Laemmli SDS buffer supplemented with 1% β-mercaptoethanol, 0.01% bromophenol blue and protease inhibitor cocktail (Roche). Equal amounts of protein were analyzed by SDS-PAGE. Proteins were transferred to PVDF membranes and incubated with primary and secondary antibodies following the manufacturers’ instructions. Primary antibodies against ASC (1:2000, AL177), caspase-1 (1:1000, Bally-1) and NLRP3 (1:1000, Cryo-2) were from Adipogen. Anti-IL-1β (1:500, AF201) antibody was from R&D. Anti-GAPDH and anti-Actin antibodies (1:5000) were from Cell Signaling. Secondary HRP-linked antibodies against rabbit and mouse IgG (1:2000) were from Cell Signaling and against goat IgG (1:2000) was from R&D. Immunoblots were revealed using the enhanced chemiluminescence reagent (Thermo Scientific) and visualized using a BIORAD camera (Universal Hood II). GAPDH or Actin was used as loading controls.

Awad F., Assrawi E., Jumeau C., Georgin-Lavialle S., Cobret L., Duquesnoy P., Piterboth W., Thomas L., Stankovic-Stojanovic K., Louvrier C., Giurgea I., Grateau G., Amselem S, & Karabina S.A. (2017). Impact of human monocyte and macrophage polarization on NLR expression and NLRP3 inflammasome activation. PLoS ONE, 12(4), e0175336.

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Immunoblot Analysis of NLRP3 Inflammasome

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Anti-β-actin (1:10,000, BH10D10) was bought from Cell Signaling Technology (Danvers, MA, United States); Anti-NLRP3 (1:1,000. Cryo-2) and Anti-ASC (1:1,000, AL177) were purchased from Adipogen (San Diego, CA, United States); Anti-Caspase-1 (1:1,000, ab179515) and Anti-NEK7 (1:10,000 ab133514) were bought from Abcam (Cambridge, CB2 0AX, United Kingdom); Anti-IL-1β (1:000 AF-401-NA; RRID: AB_416684) was obtained from RD systems (Tustin, CA, United States); the DyLight 488-labeled secondary antibody (1:50, A120-100D2) was purchased from InvivoGen (San Diego, CA, United States); and FITC anti-mouse/human CD11b (101216, 1:500 for flow cytometry) and APC anti-mouse Ly-6G (127614, 1:500 for flow cytometry) were from BioLegend.

Shi J., Xia Y., Wang H., Yi Z., Zhang R, & Zhang X. (2022). Piperlongumine Is an NLRP3 Inhibitor With Anti-inflammatory Activity. Frontiers in Pharmacology, 12, 818326.

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