HUVEC were washed with PBS according to the procedure outlined in Section 2.4 . Subsequently, 1 × 105 U937 monocytes (ATCC® CRL-1593.2TM) were added to HUVEC and incubated at 37 °C for 30 min. Following the 30 min incubation period, the unattached U937 monocytes were washed away using PBS. Under an inverted light microscope, the U937 cells that adhered to the surface of HUVEC were recognized and calculated [29 (link)].
U937 monocytes
Manufactured by American Type Culture Collection
Sourced in United States
U937 monocytes are a human monocyte cell line derived from a patient with histiocytic lymphoma. They are commonly used in research for studying monocyte and macrophage function.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (7 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (5 mentions)
Huvecs, by Thermo Fisher Scientific (5 mentions)
U937 monocytes, by Thermo Fisher Scientific (4 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Endothelial cell growth supplement (ecgs), by BD (3 mentions)
Heparin, by Merck Group (3 mentions)
Huvecs, by American Type Culture Collection (3 mentions)
Co2 incubator, by Thermo Fisher Scientific (2 mentions)
Human endothelial sfm, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin glutamine (psg), by Merck Group (1 mentions)
Human umbilical vein endothelial cells (huvec), by American Type Culture Collection (1 mentions)
Ht1080 human fibrosarcoma cells, by American Type Culture Collection (1 mentions)
Hek293t cell, by American Type Culture Collection (1 mentions)
Hela cell, by American Type Culture Collection (1 mentions)
Human endothelial serum free medium, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Egm 2 bulletkit growth media, by Lonza (1 mentions)
Lyovec transfection reagent, by InvivoGen (1 mentions)
10 protocols using u937 monocytes
1
Monocyte Adhesion to HUVEC Assay
2
Culturing Various Cell Lines for Research
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The following cell types were used: Burkitt lymphoma B-cell lines Daudi and Namalwa (ATCC, Manassas, VA), EBV-negative BL41 (BL41 −) and EBV-positive BL41 (BL4 +) (a kind gift from Bill Sugden, UW, Madison), BCBL-1, JSC-1,15 (link), PBMCs (IQ Biosciences), BC-2, HUVEC, and U937 monocytes (ATCC). Cell lines were grown in RPMI 1640 medium (Invitrogen, Carlsbad, CA) supplemented with 15% fetal bovine serum (FBS) (Thermo Scientific, Rockford, IL), 1% penicillin/streptomycin glutamine (Sigma, St. Louis, MO) at 37 °C with 5% CO2.
3
Cell Culture and Characterization of Primary and Immortalized Human Cells
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HBMVECs (10HU-051, iXCells Biotechnologies) were cultured in recommended Endothelial Cell Growth Medium (MD-0010, iXCells Biotechnologies). Cells are collected with 0.05% trypsin-EDTA and subcultured with a plating density of 5 × 103 cells/cm2. Only early passages of HBMVECs (passage 1–6) are used in our assays. U937 monocytes (ATCC) were maintained in RPMI-1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin/streptomycin (Gibco). Human microglia cell line HMC3 (CRL-3304, ATCC) was cultured in Eagle's Minimum Essential Medium (EMEM, 30–2003, ATCC), supplemented with 10% FBS (Gibco) and 1% penicillin/streptomycin (Gibco). All the cells were cultured in a 37°C incubator with 5% CO2. These cell lines have been authenticated with the short tandem repeats (STR) profiling and mycoplasma testing.
4
L. pneumophila strains and host cells
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All L. pneumophila strains are listed in Table S10 and were derived from the parent strain L. pneumophila Philadelphia-1 Lp02 (thyA hsdR rpsL). Host cell lines included U937 monocytes (ATCC CRL-1593.2) which were differentiated into macrophages with 12-O-tetradecanoylphorbol-13-acetate (TPA, Sigma-Aldrich P1585) and A. castellanii (ATCC 30234).
5
Legionella Pneumophila Infection Dynamics
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All the strains used in this study are listed in Table S1. L. pneumophila strain Lp01 (hsdR rpsL) and Lp01ΔdotA (T4SS-) are strains derived from L. pneumophila strain Philadelphia-1 (Berger and Isberg, 1993) and were cultured as previously described (Morris et al., 1979) . HEK293T cells (ATCC CRL-1573) and HT1080 human fibrosarcoma cells (ATCC CCL-121) were cultured in DMEM supplemented with 2 mM L-glutamine and 10% FBS, and incubated in 5% CO 2 at 37°C. HeLa cells (ATCC CCL-2) and U937 monocytes (ATCC CRL-1593.2) were cultured in RPMI 1640 and is also made available for use under a CC0 license.
was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105
The copyright holder for this preprint (which this version posted July 9, 2021.
was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105
The copyright holder for this preprint (which this version posted July 9, 2021.
6
Endothelial Cells and Monocyte Co-culture
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The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the Institutional Medical Ethics Committee of the Third Affiliated Hospital of Sun Yat-sen University.
The human umbilical vein endothelial cells (HUVECs) and U937 monocytes used in this study were both purchased from American Type Culture Collection (Manassas, VA, USA). HUVECs were cultured in human endothelial serum-free medium (Invitrogen, Carlsbad, CA, USA) with 20% fetal bovine serum (Invitrogen), 100 U/ml penicillin-streptomycin (Invitrogen), 100 μg/ml heparin (Sigma-Aldrich, St. Louis, MO, USA), and 150 μg/ml endothelial cell growth supplement (Becton, Dickinson and Company, Frankin Lakes, NJ, USA). U937 monocytes were cultured in RPMI1640 medium (Invitrogen), which contains 20% fetal bovine serum (Invitrogen) and 100 U/ml penicillin-streptomycin (Invitrogen). HUVECs and U937 monocytes were both cultured in an incubator (5% CO2, 37 °C, and 90% humidity) (Thermo Fisher Scientific, Waltham, MA, USA).
The human umbilical vein endothelial cells (HUVECs) and U937 monocytes used in this study were both purchased from American Type Culture Collection (Manassas, VA, USA). HUVECs were cultured in human endothelial serum-free medium (Invitrogen, Carlsbad, CA, USA) with 20% fetal bovine serum (Invitrogen), 100 U/ml penicillin-streptomycin (Invitrogen), 100 μg/ml heparin (Sigma-Aldrich, St. Louis, MO, USA), and 150 μg/ml endothelial cell growth supplement (Becton, Dickinson and Company, Frankin Lakes, NJ, USA). U937 monocytes were cultured in RPMI1640 medium (Invitrogen), which contains 20% fetal bovine serum (Invitrogen) and 100 U/ml penicillin-streptomycin (Invitrogen). HUVECs and U937 monocytes were both cultured in an incubator (5% CO2, 37 °C, and 90% humidity) (Thermo Fisher Scientific, Waltham, MA, USA).
7
Monocyte-Endothelial Cell Co-Culture Protocol
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The present study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki with the approval of the Institutional Medical Ethics Committee of the Third Affiliated Hospital of Sun Yat-sen University. Both U937 monocytes and HUVECs are obtained from American Type Culture Collection (Manassas, VA, USA). HUVECs are cultured with human endothelial SFM (Invitrogen, Carlsbad, CA, USA), containing 20% fetal bovine serum (Invitrogen), 100 U/ml penicillin-streptomycin (Invitrogen), 100 μg/ml heparin (Sigma-Aldrich, St. Louis, MO, USA), and 150 μg/ml endothelial cell growth supplement (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). U937 monocytes are cultured in RPMI1640 medium (Invitrogen), containing 20% fetal bovine serum (Invitrogen) and 100 U/ml penicillin-streptomycin (Invitrogen). These two cell lines are both cultured in a 5% CO2 incubator (37°C and 90% humidity) (Thermo Fisher Scientific, Waltham, MA, USA).
8
HIV Infection Model in U-937 and U1 Cells
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The U-937 monocytes (American Type Culture Collection) and promonocytic U1 latent cells (AIDS Research and Reference Reagent Program, NIAID, National Institute of Health, Rockville, MD, catalog no. 165) were used to perform active and latent infection model, respectively. U1 is a subclone of U937 that has been chronically infected with HIV-1, and show minimal constitutive expression of virus. The U-937 monocytes were infected with 100 ng/ml HIV-1Ba-L(NIH AIDS Research and Reference Reagent Program catalog no. 510). After 5 days of infection, RNA were isolated using the RNeasy mini kit (Qiagen, Limburg, the Netherlands) according to the manufacturer’s guidelines.
9
Culturing U937 Monocytes and HUVECs
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The present study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki with the approval of the Institutional Medical Ethics Committee of the Third Affiliated Hospital of Sun Yat-sen University. The cell lines of U937 monocytes and HUVECs are both purchased from the American Type Culture Collection (Manassas, VA, USA). U937 monocytes are cultured in RPMI1640 medium (Invitrogen, Carlsbad, CA, USA) with 20% fetal bovine serum (Invitrogen). HUVECs are cultured with human endothelial SFM (Invitrogen) with 20% fetal bovine serum (Invitrogen), 100 μg/ml heparin (Sigma-Aldrich, St. Louis, MO, USA), and 200 μg/ml endothelial cell growth supplement (Becton, Dickinson and Company, Frankin Lakes, NJ, USA). The 3 to 5 passages are used for experiments. Both U937 monocytes and HUVECs are cultured in a 5% CO2 incubator at 37°C and 90% humidity (Thermo Fisher Scientific, Waltham, MA, USA).
10
Activation of IRF3-driven Responses in Primary Cells
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Primary human cells were obtained from Lonza as single-donor aliquots. HUVEC and aortic endothelial cells were maintained in EGM-2 BulletKit growth media (Lonza) supplemented with 2% hi-FBS (fetal bovine serum). Dermal microvascular endothelial cells, brain microvascular endothelial cells, and kidney microvascular endothelial cells were maintained in EBM-2MV BulletKit growth media (Lonza) supplemented with hi-FBS. Normal bronchial epithelial cells were maintained in BEGM BulletKit growth media (Lonza). U937 monocytes were obtained from American Type Culture Collection and maintained in RPMI supplemented with hi-FBS. Purity of cell populations was verified >95% by flow cytometry using the following markers: CD31 for endothelial cells (BioLegend, 303121), epithelial cell adhesion molecule for epithelial cells (BioLegend, 324207), and CD45 for U937 monocytes (BioLegend, 103115). For activation of IRF3-driven responses, cells were transfected with poly dA:dT for 3 hours (200 ng/ml) using LyoVec transfection reagent (Invivogen) or infected for 3 hours with hCMV strain MOLD [multiplicity of infection (MOI) = 1]. For IFN-β neutralizing experiments, cells were treated with anti–IFN-β–neutralizing antibody (10 to 40 U/ml) for 72 hours.
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