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Af3625

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AF3625 is a lab equipment product manufactured by R&D Systems. It is designed to perform a specific function within a laboratory setting. The core function of this product is to facilitate a particular experimental or analytical process, but a detailed description cannot be provided while maintaining an unbiased and factual approach.

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Lab products found in correlation

Af3626, by R&D Systems (6 mentions) Biotinylated goat anti rabbit igg, by Vector Laboratories (4 mentions) Paraformaldehyde (pfa), by Thermo Fisher Scientific (2 mentions) Ezprep buffer, by Roche (2 mentions) Cc1 buffer, by Roche (2 mentions) Avidin biotin blocking, by Roche (2 mentions) Dab detection kit, by Roche (2 mentions) Permount, by Thermo Fisher Scientific (2 mentions) Baf3625, by R&D Systems (2 mentions) Hpa034881, by Merck Group (1 mentions) Ab15580, by Abcam (1 mentions) Mab424, by Merck Group (1 mentions) Sc 8431, by Santa Cruz Biotechnology (1 mentions) Phospho histone h3, by Cell Signaling Technology (1 mentions) Prb 155p, by Fortrea (1 mentions) Ab24647, by Abcam (1 mentions) Aperio versa slide scanner, by Leica (1 mentions) High ph buffer, by Vector Laboratories (1 mentions) In situ cell death detection kit, by Roche (1 mentions) Dy3626, by R&D Systems (1 mentions)

16 protocols using af3625

1

Immunostaining Protocol for Epithelial Markers

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Mouse monoclonal antibody against IL-33 (clone Nessy-1) (#ALX-804-840-C100) was purchased from Enzo. Rabbit polyclonal antibodies against KRT5 (#ab24647) and Ki-67 (#ab15580) were purchased from Abcam (Abcam, Cambridge, MA). Rabbit polyclonal antibody against KRT14 (#PRB-155P) was purchased from Covance (Covance, Princeton, NJ). Rabbit polyclonal antibody against KRT4 (#HPA034881) was purchased from Sigma (Sigma-Aldrich Corp, St. Louis, MO). Rabbit monoclonal antibodies against E-cadherin (#3195), p75 (#8238), and phospho-histone H3 (#3377) were purchased from Cell Signaling (Cell Signaling Technology, MA). Mouse monoclonal antibody against p63 (#sc-8431) was purchased from Santa Cruz (Santa Cruz Biotechnology, TX). Mouse monoclonal antibody against PCNA (#MAB424) was purchased from Millipore (Billerica, MA). Goat polyclonal antibody against IL-33 (#AF3625) was purchased from R&D (R&D Systems, Minneapolis, MN). Donkey anti-goat Alexa Fluor 488 (A11055), anti-rabbit Alexa Fluor 568 (A10042), and anti-mouse Alexa Fluor 647 (A31571) secondary antibodies were purchased from Life Technologies (Carlsbad, CA). Mouse anti-HSP90 (TA500494) and mouse anti-GAPDH (TA310153) primary antibodies were purchased from Origene (Rockville, MD).
Travers J., Rochman M., Caldwell J.M., Besse J.A., Miracle C.E, & Rothenberg M.E. (2017). IL-33 is induced in undifferentiated, non-dividing esophageal epithelial cells in eosinophilic esophagitis. Scientific Reports, 7, 17563.
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2

Immunohistochemical Analysis of IL33 Expression

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Tissues were fixed in paraformaldehyde (Fisher Scientific) for 24 hours and embedded in paraffin. The tissue sections were deparaffinized with EZPrep buffer (Ventana Medical Systems), then antigen retrieval was performed with CC1 buffer (Ventana Medical Systems). Sections were blocked for 30 minutes with Background Buster solution (Innovex), followed by avidin-biotin blocking for 8 minutes (Ventana Medical Systems). Mouse IL33 (AF3626, R&D Systems), mouse smooth muscle actin (Abcam), and human IL33 (AF3625, R&D Systems) antibodies were applied, and sections were incubated for 4 hours, followed by a 60-minute incubation with biotinylated rabbit anti-goat IgG (Vector labs), or biotinylated goat anti-rabbit IgG (Vector labs) at 1:200 dilution. Detection was performed with DAB detection kit (Ventana Medical Systems) according to the manufacturer’s instructions. Any section containing cells demonstrating cytoplasmic or nuclear positivity for IL33 was designated to have positive staining. Slides were counterstained with Masson’s trichrome, or hematoxylin, and eosin,and cover-slipped with Permount (Fisher Scientific).All histologic sections were evaluated by an independent PDAC pathologist.
Moral J.A., Leung J., Rojas L.A., Ruan J., Zhao J., Sethna Z., Ramnarain A., Gasmi B., Gururajan M., Redmond D., Askan G., Bhanot U., Elyada E., Park Y., Tuveson D.A., Gönen M., Leach S.D., Wolchok J.D., DeMatteo R.P., Merghoub T, & Balachandran V.P. (2020). ILC2s amplify PD-1 blockade by activating tissue-specific cancer immunity. Nature, 579(7797), 130-135.
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3

Quantifying IL-33+ Cells in Metastases

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Human patient samples were collected with written informed consent and processed at the Sheba Medical Center, Israel, in accordance with recognized ethical guidelines, under an approved Institutional Review Board (IRB) (3112-16). Tissue sections stained for IL-33 (AF3625, R&D systems) were analyzed by an expert pathologist. Images were scanned at X20 magnification using the Leica Aperio VERSA slide scanner. Analysis was performed using ImageScope software. In each slide, 5-10 areas of metastases and 5-10 normal areas were arbitrarily selected, and quantified for IL-33+ cells.
Shani O., Vorobyov T., Monteran L., Lavie D., Cohen N., Raz Y., Tsarfaty G., Avivi C., Barshack I, & Erez N. (2020). Fibroblast-derived IL-33 facilitates breast cancer metastasis by modifying the immune microenvironment and driving type-2 immunity. Cancer research, 80(23), 5317-5329.
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4

Quantification of IL-33 by ELISA and Luminex

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IL-33 antibodies were isolated by phage display (IL330004, IL330425, 640050) or purchased from commercial suppliers (R&D Systems #AF3625, #AF3626). IL-33 was quantified by Duoset ELISA (R&D Systems #DY3625, #DY3626), Luminex assay (Millipore #HTH17MAG-14K), according to manufacturer’s instructions, or in house ELISAs where IL330425 (150 μg ml−1), IL330004 (150 μg ml−1) or 640050 (2 μg ml−1) were used as capture antibodies. Captured IL-33 was detected with biotinylated sST2.Fc (R&D Systems #523-ST-100, 1 μg ml−1), biotinylated IL330425 (1 μg ml−1) or biotinylated goat polyclonal (R&D Systems #BAF3625, 500 ng ml−1), respectively.
Cohen E.S., Scott I.C., Majithiya J.B., Rapley L., Kemp B.P., England E., Rees D.G., Overed-Sayer C.L., Woods J., Bond N.J., Veyssier C.S., Embrey K.J., Sims D.A., Snaith M.R., Vousden K.A., Strain M.D., Chan D.T., Carmen S., Huntington C.E., Flavell L., Xu J., Popovic B., Brightling C.E., Vaughan T.J., Butler R., Lowe D.C., Higazi D.R., Corkill D.J., May R.D., Sleeman M.A, & Mustelin T. (2015). Oxidation of the alarmin IL-33 regulates ST2-dependent inflammation. Nature Communications, 6, 8327.
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5

In Vitro Neutralization of Human IL-33

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Example 12

Each test antibody (IgG) was evaluated for in vitro neutralizing activity against human IL-33, based on IL-33-dependent IFN-γ production by human peripheral blood mononuclear cell (PBMC) as indicator. A commercially available polyclonal anti-IL-33 antibody (AF3625, available from R&D Systems, Inc.) was used as a positive control. PBMCs were prepared and were inoculated in a 96-well microplate (2×105/well), and a recombinant human IL-12 (Wako Pure Chemical Industries, Inc.) was added (final concentration: 10 ng/mL) to the microplate. A mixture of each test antibody and a recombinant human IL-33 protein (10 ng/mL) was added to the microplate, and the resulting solution was incubated at a temperature of 37° C. for 48 hours. The culture supernatant was then collected, and the IFN-γ production level in the medium was measured with AlfaLISA™ human IFN-γ immunoassay kit (PerkinElmer Inc.) to evaluate IL-33 neutralizing activity. In this evaluation system, when the alkaline-purified antibodies were allowed to act at a final concentration of 10 μg/mL, the inhibition percentages were as follows: A10-1C04 exhibited 96.9% inhibition, A23-1A05 shows 97.5% inhibition, A25-2C02 exhibited 98.75% inhibition, A25-3H04 exhibited 97.9% inhibition, and A26-1F02 exhibited 98.25% inhibition.

US11725049B2. Human anti-IL-33 neutralizing monoclonal antibody (2023-08-15). Mitsubishi Tanabe Pharma Corporation [JP]. Inventors: Yasuhiro Fujino [JP], Tsutomu Yoshikawa [JP], Hiroshi Ochi [JP].
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6

Immunohistochemical Analysis of IL-33 and Proliferation

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Briefly, sections were deparaffinized, rehydrated, and antigen was unmasked in a high pH buffer (Vector Labs, Burlingame, CA). Sections were stained with goat anti-human or anti-mouse IL-33 antibodies (AF3625 and AF3626, R&D Systems, Minneapolis, MN). An isotype goat polyclonal IgG negative control was used to test for the specificity of the involved antibody at matched concentration and incubation conditions (Jackson Immunoresearch, West Grove, PA). Sections were counterstained with Mayer’s hematoxylin.
Sections were also stained with anti-Ki67 (790-4286, Ventana, Tuscon, AZ) or phospho-histone H3 (9701S, Cell signaling, Danvers, MA) to quantify proliferation. Apoptotic epithelial cells were identified based on positive TUNEL staining (In Situ Cell Death Detection Kit, Roche, Indianapolis, IN).
Waddell A., Vallance J.E., Moore P.D., Hummel A.T., Wu D., Shanmukhappa S.K., Fei L., Washington M.K., Minar P., Coburn L.A., Nakae S., Wilson K.T., Denson L.A., Hogan S.P, & Rosen M.J. (2015). IL-33 signaling protects from murine oxazolone colitis by supporting intestinal epithelial function. Inflammatory bowel diseases, 21(12), 2737-2746.
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7

Quantifying IL-33 using Phage Display

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IL-33 antibodies were isolated by phage display (#H338L293, #640050, #640104-3) or purchased from commercial suppliers (R&D Systems AF3625, AF3626). IL-33 was quantified by Duoset ELISA (R&D Systems DY3625, DY3626), according to manufacturer’s instructions, or in house ELISAs where #640050 (2 μg/mL) was used as capture antibody and detected with biotinylated goat polyclonal (R&D Systems BAF3625, 500 ng/mL).
Scott I.C., Majithiya J.B., Sanden C., Thornton P., Sanders P.N., Moore T., Guscott M., Corkill D.J., Erjefält J.S, & Cohen E.S. (2018). Interleukin-33 is activated by allergen- and necrosis-associated proteolytic activities to regulate its alarmin activity during epithelial damage. Scientific Reports, 8, 3363.
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8

Isolation and Activation of Plasmacytoid Dendritic Cells

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Buffy coats from healthy donors were used for isolation of peripheral blood mononuclear cells by density-gradient centrifugation (650g, 22°C, 30 minutes) (catalog MST00T41004; Lymphosep). pDCs were obtained with the CD304 (BDCA-4/Neuropilin-1) MicroBead Kit, human, isolation kit (catalog 130-090-532; Miltenyi Biotec) at a purity greater than 95%. pDCs were cultured in RPMI-1640/l-glutamine supplemented with 10% v/v heat-inactivated FBS, 100 IU/mL penicillin, 100 μg/mL streptomycin, and 10 mm HEPES (Gibco) for up to 18 hours. Prior to stimulation with NETs (25% v/v NET-containing supernatants or cleaved IL-33–containing supernatants) and CpG-A (0.1 μM; catalog tlrl-2216; Invivogen), pDCs were pretreated with aST2L (3 μg/mL; catalog AF523; R&D) and FcR blocking reagent (catalog 130-090532; Miltenyi Biotec) to minimize any IC carryover effect and nonspecific binding of aST2L. IC SLE NET-containing supernatants were pretreated with goat anti–human IL-33 antibody (4 μg/mL; catalog AF3625; R&D) or left untreated at 37°C for 45 minutes before their administration to pDC cultures. pDCs were also pretreated with cytochalasin D (5 μg/mL; catalog C2618; Sigma-Aldrich) or chloroquine (4 μM; Plaquenil, ATC code 8P01BA02; Sanofi Aventis) for 30 minutes to block endocytosis and TLR trafficking, respectively.
Georgakis S., Gkirtzimanaki K., Papadaki G., Gakiopoulou H., Drakos E., Eloranta M.L., Makridakis M., Kontostathi G., Zoidakis J., Baira E., Rönnblom L., Boumpas D.T., Sidiropoulos P., Verginis P, & Bertsias G. (2021). NETs decorated with bioactive IL-33 infiltrate inflamed tissues and induce IFN-α production in patients with SLE. JCI Insight, 6(21), e147671.
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9

SDS-PAGE and Western Blotting Protocol

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Samples were analysed by SDS-PAGE on NuPAGE Novex 4–12% or 12% Bis-Tris mini gels (Invitrogen) with MOPS running buffer (Invitrogen) according to manufacturer’s instructions under reducing conditions. All samples were reduced by heated to 95 C for 3 min in SDS-PAGE buffer containing 2% beta-mercaptoethanol. Gels were stained with Coomassie brilliant blue G-250 based InstantBlue gel staining (Sigma). For some studies, proteins were transferred to nitrocellulose membranes (Invitrogen) and detected by Western blotting. IL-33 was detected with goat anti-IL-33 pAb (R&D systems AF3625, AF3626) at 1:1000 dilution17 (link). Calpain-1 and -2 were detected with rabbit anti-calpain-1 or -2 pAb (Abcam ab28257, ab39165) respectively at 1:1000 dilution50 (link),75 (link). Immunoreactive proteins were identified with HRP-conjugated anti-goat or anti-rabbit (R&D systems HAF109 and HAF008 respectively) and Supersignal West Femto substrate (Pierce 34095).
Scott I.C., Majithiya J.B., Sanden C., Thornton P., Sanders P.N., Moore T., Guscott M., Corkill D.J., Erjefält J.S, & Cohen E.S. (2018). Interleukin-33 is activated by allergen- and necrosis-associated proteolytic activities to regulate its alarmin activity during epithelial damage. Scientific Reports, 8, 3363.
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10

Quantifying IL-33 Levels via Western Blot

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IL-33 levels were assessed by western blotting using human IL-33 antibody (AF3625, R&D Systems, Minneapolis, MN, USA). Details are available in the online supplement.
Jeon Y.J., Gil C.H., Won J., Jo A, & Kim H.J. (2020). Symbiotic microbiome Staphylococcus aureus from human nasal mucus modulates IL-33-mediated type 2 immune responses in allergic nasal mucosa. BMC Microbiology, 20, 301.
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