Target cell lines were selected to model the anatomic site of initial particle-cell interactions following deposition in the terminal bronchioles [24 (link)]; BEAS-2B lung bronchial epithelial cells and THP-1 human monocytic cells were selected because they have been widely used for nanotoxicology assays using harmonized exposure protocols [44 (link)]. BEAS-2B immortalized human lung bronchial epithelial cells (ATCC, Manassas, VA) were cultured in BEGM media with SingleQuots supplement kit (Lonza, Basel, Switzerland) at 37 °C in 5% CO2. THP-1 human monocytic cells (ATCC, Manassas, VA) were cultured using RPMI-1640 (Gibco) with 10% heat-inactivated fetal bovine serum (FBS) (Atlanta Biologicals, Flowery Branch, GA) and 1% penicillin/streptomycin at 37 °C and 5% CO2 in 100 mm low-attachment cell cultures dishes. Prior to use, THP-1 cells were differentiated into macrophages with 10 nM phorbol 12-myristate 13-acetate (PMA) (Fisher Scientific, Agawam, MA) for 72 h. IMR-90 human lung fibroblasts, generously provided by Dr. Anatoly Zhitkovich (Brown University, Providence, RI), were maintained using DMEM high glucose (Gibco) with 10% FBS and 1% penicillin/streptomycin at 37 °C in 5% low O2 conditions and 5% CO2. Cells were collected and washed in fresh medium prior to nanomaterial exposure. All cell lines were used before passage 20.
Thp 1 human monocytic cells
Manufactured by American Type Culture Collection
Sourced in United States
THP-1 human monocytic cells are a widely used cell line derived from the blood of a patient with acute monocytic leukemia. These cells can be differentiated into macrophage-like cells and are commonly used in research studies involving the immune system and inflammation.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (6 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (5 mentions)
Penicillin, by Thermo Fisher Scientific (4 mentions)
Streptomycin, by Thermo Fisher Scientific (4 mentions)
Rpmi 1640, by Thermo Fisher Scientific (3 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (3 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (3 mentions)
Anti eea1, by Thermo Fisher Scientific (2 mentions)
B6.129 tlr2tm1kir j mice, by Jackson ImmunoResearch (2 mentions)
Sp5 inverted confocal laser microscope, by Leica (2 mentions)
Hek293t human embryonic kidney cells, by American Type Culture Collection (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
2 mercaptoethanol, by Merck Group (2 mentions)
Rpmi 1640, by Merck Group (2 mentions)
Ebm 2, by Lonza (2 mentions)
Singlequots supplement kit, by Lonza (1 mentions)
Heat inactivated fetal bovine serum, by Atlanta Biologicals (1 mentions)
Phorbol 12 myristate 13 acetate pma, by Thermo Fisher Scientific (1 mentions)
High glucose dmem, by Thermo Fisher Scientific (1 mentions)
Huvecs, by Cambrex (1 mentions)
36 protocols using thp 1 human monocytic cells
1
Cell Lines for Nanotoxicology Assays
2
Endothelial Cell-Monocyte Adhesion Assay
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Human umbilical vein endothelial cells (HUVECs, Clonetics) were cultured (105 cells/well, in 48-well plates) in complete EGM-2 medium (Clonetics) containing 20% FBS in the presence of oxLDL (50 μg/ml) and/or various concentrations of human cortistatin-14 for 24 hours. Moreover, HUVEC monolayers were extensively washed with EGM-2 medium and co-incubated with 104 calcein AM-labelled THP1 human monocytic cells (ATCC) for 1 hour. After extensive washing, the number of THP1 cells bound to HUVEC monolayer was determined in a fluorescence microscope.
3
Mouse Model of Inflammasome Activation
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Mouse experiments were conducted in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at Louisiana State University (protocol number 16–072). All animal experiments were performed in a manner to ensure minimal pain and distress. Nlrp6-/-, Asc-/-, and Caspase-1/11-/- were obtained from the Millennium Pharmaceuticals (Cambridge, MA) whereas C57Bl/6 mice were obtained from Taconic (Rensselaer, NY) and Jackson (Bar Harbor, ME ) Laboratories. THP-1 (human monocytic) cells and HL-60 (human neutrophil-like) cells were purchased from ATCC (Manassas, VA). Lysates of human healthy control tissue and pneumonic lung tissue were obtained from Novus Biologicals, CO.
4
Macrophage Differentiation and Rhinovirus Infection
5
p62 Knockout Mouse Embryonic Fibroblast Isolation
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WT p62+/+ MEF and p62−/− MEF cells were isolated from −13.5-day embryos of WT p62+/+ mice and p62−/− mice. MEF cells with passage numbers between two and five were used in the experiments. MEF cells were cultured at 37°C in DMEM (Thermo Fisher Scientific, Waltham, MA, USA), supplemented with 10% FBS, 100 units/ml of penicillin, and 100 µg/ml of streptomycin. HEK293T human embryonic kidney cells were purchased from the American Type Culture Collection (ATCC, CRL-11-268) and maintained in DMEM (Thermo Fisher Scientific). THP-1 human monocytic cells were purchased from ATCC (TIB-202) and maintained in RPMI 1640 medium (Thermo Fisher Scientific) containing 10% FBS (Hyclone, Ogden, UT, USA), 2 mM L-glutamine (Gibco, Grand Island, NY, USA), 100 units/ml penicillin (Gibco), 100μg/ml streptomycin (Gibco), and 5×10−5 M β-mercaptoethanol (Gibco).
6
Culturing Cell Lines for Biomedical Research
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HEK293T human embryonic kidney cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, Carlsbad, CA, USA). HEK293 cells expressing human TLR4 (293/TLR4) were purchased from InvivoGen (San Diego, CA, USA) and maintained in DMEM containing 4.5 g/l glucose, 2–4 mM l -glutamine, 10% fetal bovine serum (FBS), 50 U/ml penicillin, 50 µg/ml streptomycin, 100 µg/ml Normocin according to the manufacturer’s protocol. THP-1 human monocytic cells were purchased from ATCC and maintained in RPMI medium (Invitrogen) containing 10% FBS, 2 mM l -glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 5 × 10−5 M β-mercaptoethanol. Human breast carcinoma cell line MDA-MB-231 and human hepatic adenocarcinoma cell line SK-HEP-1 were obtained from ATCC and maintained in DMEM (Invitrogen) supplemented with 10% FBS. Dimethyl sulfoxide (DMSO, D8418), 3-methyladenine (3-MA, M9281), and pepstatin A (P4265) were purchased from Sigma (Sigma-Aldrich, St. Louis, MO, USA). Stock solutions were prepared in DMSO. The final concentration of DMSO in culture medium was <0.2% volume.
7
Macrophage Differentiation and Rhinovirus Infection
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To generate bone marrow-derived macrophages, mouse bone marrow monocytes were isolated from C C57BL/6J and TLR2−/− (B6.129-Tlr2tm1Kir/J) mice (Jackson Laboratories) and cultured in L929 medium, a source of macrophage colony-stimulating factor, for 7 days 49 (link). Human primary peripheral blood mononuclear cells (PBMC; Precision for Medicine, Frederick, MD) were cultured in RPMI-1640 media supplemented with 20ng/mL of recombinant human M-CSF. THP-1 human monocytic cells (ATCC) were differentiated with 5 ng/ml of phorbol 12-myristate 13-acetate as described50 . Cells were infected with RV-A1B and RV-C15 at a multiplicity of infection (MOI) of 1.0 at 33°C. To study virus entry, human THP-1-derived macrophages and mouse bone marrow-derived macrophages were incubated with RV-A1B or RV-C15 for 1 hour and washed three times with PBS. Viral RNA and CDHR3 mRNA expression were quantified by qPCR (see below) and the presence of RV-C was examined by Western blot and immunofluorescence using anti-EV-D68 vp3. Late endosomes were visualized with anti-EEA1 (Invitrogen, Carlsbad, CA). Images were visualized using a Leica SP5 inverted laser confocal microscope (Buffalo Grove, IL).
8
Epithelial and Monocytic Cell Lines
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Caco-2 human colon epithelial cells were obtained from American Tissue Culture Collection (ATCC, CAT#: HTB-37, Manassas, VA). THP-1 human monocytic cells were obtained from ATCC (CAT#: TIB-202). HT29-MTX-E12 cells, mucous-secreting human intestinal cells, were obtained from Millipore Sigma (CAT#: 12040401, Burlington, MA). Bacillus subtilis (3A1T (wild-type)) strain was obtained from Bacillus Genetic Stock Center (Ohio State University, Columbus, OH). All cell lines were authenticated by the suppliers and obtained with the appropriate Material Transfer Agreement. Mycoplasma testing of human cells gave negative results.
9
HUVEC, Jurkat, and THP-1 Cell Culture
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The RPMI-1640 media, penicillin, streptomycin, dimethyl sulfoxide (DMSO), and trypan blue (TB) were purchased from Sigma Aldrich (Mo, USA). Fetal calf serum (FCS) was purchased from Gibco (USA). MTT (3-[4, 5-dimethylthiazol-2, 5-diphenyltetrazolium bromide]) reagent was purchased from Invitrogen (USA). Cell culture microtiter plates, and tubes (Corning Falcon, USA). Human umbilical vein endothelial cells (HUVEC), Jurkat clone E6-1 cells and THP-1 human monocytic cells were purchased from American Type Culture Collection (16549; Cat no. PCS-100-010; ATTCC, Manassas, VA, USA) and (ATCC-TIB202), respectively.
10
Culturing THP-1 and Jurkat T Cells
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THP-1 human monocytic cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained at 37°C in RPMI 1640 medium supplemented with 10% FBS in the presence of penicillin and streptomycin. Jurkat T cells stably expressing CCR5 were maintained in RPMI 1640 medium supplemented with 10% FBS in the presence of geneticin (20 (link)).
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