U937 cells

Manufactured by American Type Culture Collection

Sourced in United States

U937 cells are a human monocyte-like cell line derived from a patient with diffuse histiocytic lymphoma. These cells are widely used in research to study monocyte and macrophage biology, including inflammation, cell signaling, and disease models.

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100 protocols using u937 cells

Cell Culture Protocol for Leukemia Cell Lines

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RCH-ACV and Kasumi-2 cells were purchased from DSMZ (Germany). RCH-ACV+ROR1V5, SupB15, and REH cells were described previously (10 (link)). U937 cells were purchased from ATCC. All cell lines were cultured in RPMI1640 supplemented with 10% FBS, 2% L-Glutamine, 1% penicillin/streptinomycin, and 0.1% Fungizone (R10). All cells were grown at 37°C with 5% CO₂.

Chow M., Gao L., MacManiman J.D., Bicocca V.T., Chang B.H., Alumkal J.J, & Tyner J.W. (2018). Maintenance and Pharmacologic Targeting of ROR1 Protein Levels via UHRF1 in t(1;19) pre-B-ALL. Oncogene, 37(38), 5221-5232.

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Cell Culture Conditions for Cancer Research

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NB4 cells (DSMZ), HL-60 cells (ATCC), U937 cells (ATCC), U937/PR9 (PR9) cells, and NB4-MR2 cells (kindly provided by Dr. Wilson Miller Jr., McGill University, Montreal, QC) were maintained in RPMI-1640 medium (Invitrogen) supplemented with 10% FBS and 1% penicillin/streptomycin. MCF-7, MDA-MB-231, SUM159PT cells (kindly provided by Dr. Paola Marcato, Department of Pathology, Dalhousie University, Halifax, NS) and HEK293T cells (ATCC) were maintained in DMEM (Invitrogen) supplemented with 10% FBS and 1% penicillin/streptomycin. Non-adherent cells were maintained with the cell density kept at < 1 × 10⁶ cells/mL.

Holloway R.W., Thomas M.L., Cohen A.M., Bharadwaj A.G., Rahman M., Marcato P., Marignani P.A, & Waisman D.M. (2018). Regulation of cell surface protease receptor S100A10 by retinoic acid therapy in acute promyelocytic leukemia (APL)☆. Cell Death & Disease, 9(9), 920.

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Cell Culture Protocols for U937, HEK293T, and Primary Human Monocytes

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U937 cells (CRL‐1593.2) were purchased from the ATCC. The cells were cultured according to the manufacturer's instructions, in RPMI‐1640 medium (Life Technologies) supplemented with 100 U/ml penicillin (GIBCO), 100 μg/ml streptomycin (GIBCO), and 10% (v/v) heat‐inactivated fetal bovine serum (GIBCO; complete RPMI medium). The cells were incubated at 37°C under 5% CO₂.
HEK293T cells (CRL‐3216) were purchased from ATCC. The cells were cultured according to the manufacturer's instructions, in DMEM (Life Technologies) supplemented with 100 U/ml penicillin (GIBCO), 100 μg/ml streptomycin (GIBCO), 1× Glutamax (GIBCO), and 10% heat‐inactivated fetal bovine serum (complete DMEM medium). The cells were incubated at 37°C under 5% CO₂.
Primary human monocytes were obtained by culturing primary human monocytes enriched from buffy coats as described previously (Rieckmann et al,2017 (link)). Primary human macrophages were differentiated in RPMI‐1640 medium (Life Technologies) supplemented with 100 U/ml penicillin (GIBCO), 100 μg/ml streptomycin (GIBCO), 10% (v/v) heat‐inactivated fetal bovine serum (GIBCO), and 50 ng/ml M‐Csf. The cells were incubated at 37°C under 5% CO₂.

Frauenstein A., Ebner S., Hansen F.M., Sinha A., Phulphagar K., Swatek K., Hornburg D., Mann M, & Meissner F. (2021). Identification of covalent modifications regulating immune signaling complex composition and phenotype. Molecular Systems Biology, 17(7), e10125.

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Endothelial Cell Migration Assay

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ECs from pooled human umbilical cords were cultured in M199 medium supplemented with 20% FBS, 0.1% heparin, 50 μg/mL ECGF (Biomedical Technologies), penicillin/streptomycin on gelatin coated tissue culture plates. U937 cells (ATCC) were maintained in RPMI with 10% FBS and antibiotics. For trans-endothelial migration (see below), HUVEC (subculture 2) were grown on fibronectin-coated glass coverslips (5 mg/mL; BD Biosciences) and treated with JQ1 (500 nM) or vehicle (DMSO) for 1 hour before TNFα stimulation (10 ng/mL, 4 hours). Recombinant human TNFα was obtained from PeproTech (Rocky Hill, NJ). JQ1 was dissolved in DMSO at a concentration of 50 mg/mL. Working stocks of JQ1 were prepared by diluting 1:10 in 10% beta-cyclodextrin solution (Filippakopoulos et al., 2010 (link)). Animals were treated at 50 mg/kg once daily by intraperitoneal injection

Brown J.D., Lin C.Y., Duan Q., Griffin G., Federation A., Paranal R.M., Bair S., Newton G., Lichtman A., Kung A., Yang T., Wang H., Luscinskas F.W., Croce K., Bradner J.E, & Plutzky J. (2014). NF-kB directs dynamic super enhancer formation in inflammation and atherogenesis. Molecular cell, 56(2), 219-231.

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Monocyte Adhesion to Adipocytes

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Differentiated 3T3-L1 adipocytes were cultured for 7 days in 5 mmol/L or 25 mmol/L glucose. To examine how many monocytes attached to the cultured adipocytes, U937 cells (10⁶ cells /mL, ATCC) were fluorescently labeled by incubation with calcein-AM (5 μg/mL, Molecular Probes) for 30–45 minutes, and washed twice in phenol red-free RPMI medium (GIBCO) (Yeop Han et al., 2010 (link)). Suspended U937 cells were then added to 3T3-L1 adipocyte monolayers and allowed to adhere for 90 minutes at 4°C. Plates were washed gently three times and visualized to ensure monolayer integrity, before measuring fluorescence in a FusionTM Series Universal Microplate Analyzer (Packard Bioscience) with excitation and detection wavelengths of 485 and 535 nm, respectively.

Han C.Y., Kang I., Harten I.A., Gebe J.A., Chan C.K., Omer M., Alonge K.M., den Hartigh L.J., Kjerulf D.G., Goodspeed L., Subramanian S., Wang S., Kim F., Birk D.E., Wight T.N, & Chait A. (2020). Adipocyte-Derived Versican and Macrophage-Derived Biglycan Control Adipose Tissue Inflammation in Obesity. Cell reports, 31(13), 107818.

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Characterization of Dengue Virus Strains

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Vero cells (ATCC No CCL-81) were grown at 37°C in a humidified 5%-CO₂ incubator in medium 199 with Earle's salts supplemented with 5%-FBS (Gibco). U937 cells (ATCC No CRL-1593.2) were grown at 37°C in a humidified 5%-CO₂ incubator in RPMI 1640 medium supplemented with 10%-FBS (Gibco). The DENV-1 0111/2011 and DENV-2 0126/2010 strains [23 (link)] were kindly provided by Dr Ana Bispo de Filippis (Instituto Oswaldo Cruz, Rio de Janeiro, Brazil), and used both for in vivo viral challenge and plaque-reduction neutralization test (PRNT). The DENV-1 WestPac-74 and DENV-2 S16803 strains were kindly provided by Dr Kenneth Eckels (Walter Reed Army Institute of Research, Silver Spring, USA), and used for in vivo viral challenge, PRNT, and ADE assay (DENV-2 S16803), and for ADE assay (DENV-1 WestPac-74). The DENV-1 60305 [74 (link)], DENV-2 44/2 [75 ], DENV-3 16562 [74 (link)] and DENV-4 TVP360 strains were used in PRNT.

Borges M.B., Marchevsky R.S., Carvalho Pereira R., da Silva Mendes Y., Almeida Mendes L.G., Diniz-Mendes L., Cruz M.A., Tahmaoui O., Baudart S., Freire M., Homma A., Schneider-Ohrum K., Vaughn D.W., Vanloubbeeck Y., Lorin C., Malice M.P., Caride E, & Warter L. (2019). Detection of post-vaccination enhanced dengue virus infection in macaques: An improved model for early assessment of dengue vaccines. PLoS Pathogens, 15(4), e1007721.

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Cell Line Establishment and Characterization

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Human LPS cell lines Lipo224, Lipo246, Lipo863 were established in our laboratory as previously reported (11 (link)). LPS141 was kindly provided by Dr. Jonathan Fletcher (Brigham and Women’s Hospital). LPS cells were maintained using standard conditions and were grown in DMEM (Gibco), supplemented with 10% (vol/vol) FBS. U937 cells (ATCC) were cultured in RPMI (Gibco), supplemented with 10% (vol/vol) FBS. For differentiation, cells were incubated with 12-myristate 13-acetate (PMA) 10 ng/ml for 24h. Human HEK-Blue-293 (Invivogen) cells were cultured in DMEM supplemented with 10% (vol/vol) FBS, Normocin (50 μg/mL), Blasticidin (10 μg/mL), and Zeocin (100 μg/mL) (Invivogen). Preadipocytes (XA15A1) were purchased from Lonza and maintained following the manufacturer’s instructions. Murine Lewis lung carcinoma cells (LLC) (ATCC) were cultured in RPMI 1640, supplemented with 10% (vol/vol) FBS.
All the cell line used in this study were acquired within the past 5 years and authenticated by STR on 2/9/2017. Murine Lewis lung carcinoma cells (LLC) were authenticated by morphology and biologic behavior. Preadipocytes and HEKBlue 293 were bought within 6 month from this manuscript submission. Lonza tested the cells for differentiation, and stained for adipocytes; HEK-Blue-293 were thoroughly tested and validated by InvivoGen. All cell lines were tested for mycoplasma.

Casadei L., Calore F., Creighton C.J., Guescini M., Batte K., Iwenofu O.H., Zewdu A., Braggio D.A., Lynn Bill K., Fadda P., Lovat F., Lopez G., Gasparini P., Chen J.L., Kladney R.D., Leone G., Lev D., Croce C.M, & Pollock R.E. (2017). Exosome-derived miR-25-3p and miR-92a-3p stimulate liposarcoma progression. Cancer research, 77(14), 3846-3856.

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Assessing U937 Cell Binding to HLF-Secreted ECM

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Subsets of cocultured HLFs were used to assess the ability of the secreted ECM to bind U937 cells (ATCC). Cells were washed twice in phenol-free media and resuspended (3 × 10⁶ cells/ml). HLF wells were washed with RPMI. Following this, 1.0 ml of the leukocyte suspension was added to the wells and allowed to bind at 4 °C for 90 min. Cultures were washed five times in cold RPMI to remove nonadherent cells. U937 cell binding was imaged with fluorescent microscopy following fixation on coverslips. Fixed U937 cell binding assays followed the same IHC protocol as above for HABP staining with the modification of a primary antibody against the monocyte marker CD68 (1:200, mouse KP-1 monoclonal, Abcam) and a secondary antibody (1:1000, donkey anti-mouse Alexa Fluor 488, Thermo Fisher) at the corresponding steps in the staining protocol. Cell counts were performed using ImageJ software (NIH, Bethesda, MD).

Kellar G.G., Barrow K.A., Rich L.M., Debley J.S., Wight T.N., Ziegler S.F, & Reeves S.R. (2020). Loss of versican and production of hyaluronan in lung epithelial cells are associated with airway inflammation during RSV infection. The Journal of Biological Chemistry, 296, 100076.

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Immunomodulation of U937 Monocytes on Scaffolds

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Human monocytes (U937 cells) procured from ATCC (Manassas, VA, USA) were utilized for immunomodulation study at passage 17 to 19. The cells were cultured in RPMI 1640 media with 1% antibiotic and 10% fetal bovine serum obtained from Gibco Life Technologies (Grand Island, NY, USA). Media was replaced every alternate day. For experiments, 1 × 10⁵ U937 cells were seeded on each prepared scaffold and cultured for 48 hour (h). For control, cells were cultured on tissue culture polystyrene surface (TCPS) and for the positive control, the cells were treated with 15 nM PMA before seeding.

Rather H.A., Varghese J.F., Dhimmar B., Yadav U.C, & Vasita R. (2022). Polycaprolactone-collagen nanofibers loaded with dexamethasone and simvastatin as an osteoinductive and immunocompatible scaffold for bone regeneration applications. Biomaterials and Biosystems, 8, 100064.

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Monocyte Adhesion to Endothelial Cells

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Monocyte adhesion to ECs was determined using U937 cells (ATCC, Manassas, VA, USA) as previously described by our group [27] (link). In brief, HUVECs were grown to confluence in 96-well plates. After treatment, the HUVECs were gently washed with serum-free media, and calcein-AM-labeled U937 cells (5×10⁴/ml DMEM medium containing 1% FBS) were then added. After incubation for 1 h, the HUVEC monolayer was gently washed with phosphate-buffered saline (PBS) to remove unbound monocytes. The fluorescence was measured to determine the levels of bound monocytes using a microplate reader (SpectraMax 190, Molecular Device, USA) at excitation and emission wavelengths of 496 and 520 nm, respectively.

Wang G., Liu K., Li Y., Yi W., Yang Y., Zhao D., Fan C., Yang H., Geng T., Xing J., Zhang Y., Tan S, & Yi D. (2014). Endoplasmic Reticulum Stress Mediates the Anti-Inflammatory Effect of Ethyl Pyruvate in Endothelial Cells. PLoS ONE, 9(12), e113983.

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