Ab120297

Manufactured by Abcam

Sourced in United Kingdom

Ab120297 is a monoclonal antibody that binds to the protein Galectin-3. It can be used for various research applications, including immunohistochemistry, immunocytochemistry, and western blotting.

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Lab products found in correlation

Ab120499, by Abcam (3 mentions) Ionomycin calcium salt, by Thermo Fisher Scientific (2 mentions) Ab141574, by Abcam (1 mentions) Bay 11 7805, by Abcam (1 mentions) Cd11b, by BioLegend (1 mentions) Ary005b, by R&D Systems (1 mentions) Elisa kit, by BD (1 mentions) Srp3274, by Merck Group (1 mentions) Ifn γ, by Novoprotein (1 mentions) Monensin sodium salt, by Abcam (1 mentions) Flow cytometry antibodies, by BioLegend (1 mentions) Ab270079, by Abcam (1 mentions) Human ifn γ elispot set, by BD (1 mentions) Cholecalciferol, by Merck Group (1 mentions)

13 protocols using ab120297

Mechanistic Insights into HIBD Pathways

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Besides the sham group (n = 10) and the HIBD group (n = 10), the remaining successfully modeled rats were randomly divided into the following groups:
Phorbol-12-myristate-13-acetate (PMA) group: PMA (ab120297, Abcam Inc., Cambridge, MA, USA) was dissolved in dimethyl sulfoxide (DMSO) with the final concentration 100 mM and intraperitoneally injected into the HIBD rats (n = 10);
BAY-11–7805 group: The NF-κB inhibitor BAY-11–7805 (ab141574, Abcam Inc., Cambridge, MA, USA) was dissolved in DMSO with the final concentration 10 μM and injected intraperitoneally into the HIBD rats (n = 10);
si-RCAN1 group: RCAN1 AAV siRNA (iAAV05614009, ABM Inc., Richmond, BC, Canada) packaged viruses were injected into the left hippocampus of successfully modeled HIBD rats by stereo injection (n = 10);
PMA + si-RCAN1 group: PMA and RCAN1 AAV siRNA packaged viruses were injected stereoscopically into the successfully modeled HIBD rats (n = 10).

Fang H., Li H.F., Yang M., Liao R., Wang R.R., Wang Q.Y., Zheng P.C., Zhang F.X, & Zhang J.P. (2019). NF-κB signaling pathway inhibition suppresses hippocampal neuronal apoptosis and cognitive impairment via RCAN1 in neonatal rats with hypoxic-ischemic brain damage. Cell Cycle, 18(9), 1001-1018.

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Differentiation of U937 Monocytes into Macrophages

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U937 cells were cultured in the suggested medium for 24 h before priming. U937 monocytes were primed with 80 ng/ml phorbol 12‐myristate‐13‐acetate (PMA) (Abcam, ab120297) for 48 h to induce the formation of monocyte‐derived macrophages.¹⁴ Then, flow cytometry was used to verify whether U937 cells were polarized into macrophages by detecting the expression of CD68 (BioLegend, 564944) and CD11b (BioLegend, 301345), the markers of macrophages. All assays were performed in triplicate.

Miao Y., Deng Y., Liu J., Wang J., Hu B., Hao S., Wang H., Zhang Z., Jin Z., Zhang Y., Li C., Zhang P., Wan H., Zhang S., Feng J, & Ji N. (2022). Anti‐cancer effect of targeting fibroblast activation protein alpha in glioblastoma through remodeling macrophage phenotype and suppressing tumor progression. CNS Neuroscience & Therapeutics, 29(3), 878-892.

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Culturing and Differentiating Cell Lines for SARS-CoV-2 Research

Cited 1 time

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Vero CCL-81 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin-streptomycin, 4.5 g/L glucose, 2 mM L-glutamine, 1 mM sodium pyruvate, and 1.5 g/L NaHCO₃. Calu-3 cells were cultured in DMEM medium supplemented with 20% FBS, 1% non-essential amino acids, 4.5 g/L glucose, 2 mM L -glutamine, 1 mM sodium pyruvate, 100 U/mL penicillin-streptomycin and 1.5 g/L NaHCO₃. THP-1 cells were cultured in RPMI-1640 supplemented with 10% FBS, and 1% penicillin-streptomycin at 37 °C. All cells were kept in a humidified 5% CO₂ atmosphere. THP-1 monocyte cells were differentiated into macrophage-like cells as described by Gatto et al. [77 (link)] with few modifications. THP-1 monocytes were induced to differentiate into macrophages by the addition of phorbol-12-myristate 13-acetate (PMA, 50 ng/mL) (ab120297, Abcam, Cambridge, UK) for 48 h (h). After this time, the PMA-containing medium was replaced with fresh medium without PMA for 24 h prior to SARS-CoV-2 infection. Cell differentiation was verified by evaluating cell adhesion and spreading under an optical microscope.

Macedo-da-Silva J., Rosa-Fernandes L., Gomes V.D., Santiago V.F., Santos D.M., Molnar C.M., Barboza B.R., de Souza E.E., Marques R.F., Boscardin S.B., Durigon E.L., Marinho C.R., Wrenger C., Marie S.K, & Palmisano G. (2023). Protein Arginylation Is Regulated during SARS-CoV-2 Infection. Viruses, 15(2), 290.

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Comprehensive Cell Surface Analysis

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For cell‐surface analysis, cells were stained with the anti‐mouse Zombie NIR Fixable Viability Kit (423106), and incubated with antibodies against CD45 (103114), CD11b (101205), F4/80 (123121), CD3 (100212), CD4 (100408), and CD8a (100752) at the recommended concentrations at 4 °C for 30 min. For the T‐cell intracellular IFN‐γ (505808) cytokine staining, cells were fixed and permeabilized after stimulation with Phorbol 12‐myristate 13‐acetate (PMA) (ab120297, Abcam, 100 ng mL⁻¹), Monensin sodium salt (ab120499, Abcam, 1 ug mL⁻¹), and Ionomycin calcium salt (5608212, PeproTech, 100 ng mL⁻¹) for 6 h. For the CD206 (141706) staining, cells were also fixed and permeabilized. All flow cytometry antibodies were purchased from Biolegend (San Diego, CA, USA).

Lu L., Zi H., Zhou J., Huang J., Deng Z., Tang Z., Li L., Shi X., Lo P., Lovell J.F., Deng D., Wan C, & Jin H. (2023). Engineered Microparticles for Treatment of Murine Brain Metastasis by Reprograming Tumor Microenvironment and Inhibiting MAPK Pathway. Advanced Science, 10(8), 2206212.

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Cell Culture Protocols for HCE, WKD, and THP-1 Cells

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Cells were obtained from ATCC (Manassas, VA, USA) and cultured under standard conditions (5% CO₂, 95% humidified air, 37 °C). Human corneal epithelial cells (HCE) cells (CRL11135) were cultured, as previously described in Gross et al. [47 (link)], in DMEM supplemented with 10% heat-inactivated FBS, 2% glutamine, 5 µg/mL insulin transferine selenium, 0.1 µg/mL cholera toxin, 1% antibiotics (ampicillin 100 U/mL, streptomycin 100 µg/mL, and amphotericin B 25 µg/mL), 10 ng/mL epithelial growth factor, and 0.5% dimethyl sulfoxide. The Wong–Kilbourne derivative of Chang conjunctival cells (WKD) (CCL-20) were cultured in Dulbecco’s modified Eagle’s Medium culture supplemented with 1% antibiotics, 2% glutamine, and 10% heat-inactivated FBS. The THP-1 cell line (TIB-202) was cultured in a RPMI 1640 culture medium supplemented with 1% antibiotics, 2% glutamine, and 10% heat-inactivated FBS. The differentiation of THP1 into macrophages was carried out using a treatment with 6 ng/mL PMA (phorbol 12-myristate 13-acetate, ab120297, Abcam, Cambridge, UK).

Henrioux F., Navel V., Belville C., Charnay C., Antoine A., Chiambaretta F., Sapin V, & Blanchon L. (2023). Inflammation of Dry Eye Syndrome: A Cellular Study of the Epithelial and Macrophagic Involvement of NFAT5 and RAGE. International Journal of Molecular Sciences, 24(13), 11052.

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Macrophage Differentiation and Secretome Analysis

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iSNAP‐MC cells were induced to differentiate into macrophages for 48 h by the treatment of 20 ng/m PMA (ab120297; Abcam). The cell conditioned medium was collected and centrifuged to remove cell debris at 1000g for 5 min. The supernatant was collected and stored in −80°C. Cell secretion profile was analyzed with a cytokine array (ARY005B; R&D). Secretions of TNFα and IL‐10 in the conditioned medium were quantified by using ELISA kits (DY210‐05 and DY217B‐05; BD Pharmingen). All values are expressed as mean ± SD and analyzed statistically by the double tailed t‐test. Significance was determined from p < 0.05.

Wu H., Amirfakhri S., Lin H., Hollandsworth H., Filemoni F., Liu Y., Wu Y., Li J.Y., Xu H., Chien S., Bouvet M, & Wang Y. (2022). Monocytes engineered with iSNAP inhibit human B‐lymphoma progression. Bioengineering & Translational Medicine, 7(2), e10285.

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Macrophage Differentiation Protocol

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THP-1 cells (1×105/well) were inoculated in the 24-well plate, followed by 24-h exposure to 100 ng/ml phorbol 12-myristate 13-acetate (PMA) (Abcam, ab120297) for differentiation induction to M0 macrophages. Then, lipopolysaccharide (100 ng/ml, LPS) (Sigma Aldrich, L2630) as well as interferon-gamma (20 ng/ml, IFN-γ) (Novoprotein, GMP-CI57) was added to induce differentiation into M1 macrophages. M0 macrophages were also exposed to interleukin (IL)-4 (20 ng/ml, Sigma Aldrich, SRP3093-20UG) together with IL-13 (20 ng/ml, Sigma Aldrich, SRP3274) for 24 h to induce differentiation into M2 macrophages.

Zhang Q., Wang X., Liu Y., Xu H, & Ye C. (2024). Pan-cancer and single-cell analyses identify CD44 as an immunotherapy response predictor and regulating macrophage polarization and tumor progression in colorectal cancer. Frontiers in Oncology, 14, 1380821.

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Multiparameter Flow Cytometry of Immune Cells

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For cell-surface analysis, cells were stained with anti-mouse Zombie Violet™ Fixable Viability Kit (423114), Zombie NIR™ Fixable Viability Kit (423106), CD45 (103114), CD11b (101205), CD86 (105012), CD4 (100408), CD8a (100752), Gr-1 (108411), and PD-L1 (124311, 124308) in recommended antibody concentrations and incubated at 4 °C for 30 min. For the T-cell intracellular IFN-γ (505808) cytokine staining, cells were fixed and permeabilized after stimulation with Phorbol 12-myristate 13-acetate (PMA) (ab120297, Abcam, 100 ng/mL), Monensin sodium salt (ab120499, Abcam, 1 ug/mL), and Ionomycin calcium salt (5608212, PeproTech, 100 ng/mL) for 6 h. For the CD206 (141706) and Foxp3 (126408) staining, cells were also fixed and permeabilized. All flow cytometry antibodies were purchased from Biolegend (San Diego, CA, USA).

Dai X., Lu L., Deng S., Meng J., Wan C., Huang J., Sun Y., Hu Y., Wu B., Wu G., Lovell J.F., Jin H, & Yang K. (2020). USP7 targeting modulates anti-tumor immune response by reprogramming Tumor-associated Macrophages in Lung Cancer. Theranostics, 10(20), 9332-9347.

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Multiparametric Flow Cytometry of Immune Cells

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Cells were stained with the anti-mouse Zombie NIR reparable survival kit (423,106) and incubated with anti-CD45(103,114), CD11B (101,205), f 4/80(123,121), CD3(100,212), CD4(100,408), and CD8A (100,752) at recommended concentrations for 30 min at 4 ° C to stain the cell surface. For intracellular IFN-γ (505,808) cytokine staining in T cells, phorbol 12-myristate 13-acetate (PMA) was used first (AB120297, ABCAM, 100 ng/mL), monensin sodium salt (AB120499, Abcam, 1 µg/mL) and ionomycin calcium salt (5,608,212, PeproTech, 100 ng/mL) stimulated T cells for 6 h, after which cells were fixed and permeabilized. For CD206(141,706) staining, cells were also immobilized and permeabilized. All Flow cytometry antibodies were purchased from BioLegend (San Diego, CA).

Deng S., Wang J., Hu Y., Sun Y., Yang X., Zhang B., Deng Y., Wei W., Zhang Z., Wen L., Qin Y., Huang F., Sheng Y., Wan C, & Yang K. (2024). Induction of therapeutic immunity and cancer eradication through biofunctionalized liposome-like nanovesicles derived from irradiated-cancer cells. Journal of Nanobiotechnology, 22, 156.

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Induction of M2 Macrophage Polarization

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The induction of M2 polarization of macrophages was performed as previously described.¹⁰, ³³ Briefly, the differentiation of THP‐1 cells into macrophages was induced by treating with phorbol 12‐myristate 13‐acetate (PMA; #ab120297; Abcam; 320 nM) at 37°C for 6 h, which were then incubated with PMA, IL‐4 (#ab155733; Abcam; 20 ng/mL), and IL‐13 (#ab270079; Abcam; 20 ng/mL) for 18 h at 37°C. The identity of M2 macrophages was confirmed by detecting the expression of M2 macrophage biomarkers including CD206, CD163, Arginase‐1 (Arg‐1), transforming growth factor β1 (TGF‐β1), and IL‐10 through flow cytometry and quantitative RT‐PCR.

Sui H., Cui X., Shi C., Yan Z., Li H, & Wang F. (2023). LRRC75A‐AS1 delivered by M2 macrophage exosomes promotes cervical cancer progression via enhancing SIX1 expression. Cancer Science, 114(6), 2634-2649.

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