The anti-inflammatory assay was performed as in Lauritano et al. [12 (link)]. Briefly, ∼106 human monocyte THP-1 cells/mL (ATCC® TIB-202TM) supplemented with 50 ng/mL phorbol 12- myristate 13-acetate (PMA, Sigma-Aldrich) were seeded in 96-well plates and incubated at 37 °C, 5% CO2 for 48 h in RPMI-1640 medium (Biochrom). After 72 h, 80 μL fresh RPMI medium and 10 μL/well of test sample were added. In particular, fractions A, B, C, D, and E were tested at 100 and 50 μg/mL, while 1-palmitoyl-sn-glycero-3-phosphocholine (Sigma L5254) was tested at 3.13, 6.25, 12.5, 25, and 50 μg/mL. The tests were performed at least in triplicate. After incubation for 1 h, all samples were incubated with 1 ng/mL lipopolysaccharide (LPS) for 6 h at 37 °C and plates were then frozen at −80 °C. Enzyme-linked immunosorbent assay (ELISA) was used to test TNF-α inhibition. ELISA was performed as in Lauritano et al. [12 (link)]. Results were read at 405 nm.
Tib 202tm
Manufactured by American Type Culture Collection
Sourced in United States
The TIB-202TM is a laboratory equipment designed for the cultivation and maintenance of cell cultures. It provides a controlled environment for the growth and propagation of various cell lines, ensuring optimal conditions for cell proliferation and viability. The TIB-202TM is a reliable tool for researchers and scientists working in the field of cell biology, tissue engineering, and related disciplines.
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Lab products found in correlation
Phorbol myristate acetate (pma), by Merck Group (3 mentions)
Phorbol 12 myristate 13 acetate pma, by Merck Group (3 mentions)
Rpmi 1640, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (3 mentions)
Lipopolysaccharides (lps), by Merck Group (3 mentions)
L glutamine, by Merck Group (2 mentions)
Penicillin streptomycin, by Merck Group (2 mentions)
Hepes, by Merck Group (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Penicillin and streptomycin, by Thermo Fisher Scientific (2 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
1 palmitoyl sn glycero 3 phosphocholine, by Merck Group (1 mentions)
Rpmi 1640 medium, by Harvard Bioscience (1 mentions)
Anti cd11c microbeads, by Miltenyi Biotec (1 mentions)
Anti calnexin, by Cell Signaling Technology (1 mentions)
Rpmi 1640 media, by Thermo Fisher Scientific (1 mentions)
Anti actin sc 47778, by Santa Cruz Biotechnology (1 mentions)
23 protocols using tib 202tm
1
Anti-Inflammatory Assay of Natural Compounds
2
Differentiation of Immune Cell Types
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Human THP-1 cells (ATCC® TIB-202TM) were differentiated to macrophages (THP-1 macrophages) with 60 nM phorbol 12-myristate 13-acetate (PMA; Sigma) for 16 h, and cells were cultured for an additional 48 h without PMA. Human monocyte-derived dendritic cells (MDDC) were isolated by stimulating isolated monocytes with granulocyte-macrophage colony-stimulating factor (GM-CSF, 1000 U/ml) and IL-4 (250 U/ml) and maintained in RPMI-1640 medium supplemented with 10% heat-inactivated FCS and 1% penicillin–streptomycin (Invitrogen-Gibco)35 (link),56 (link),57 (link). Bone marrow cells were isolated from the tibia and femur and cultured in RPMI1640 medium with 10% FBS, 1% penicillin–streptomycin, and 10% L929 conditioned media containing macrophage-colony stimulating factor (M-CSF) for 6 days, 25 ng/ml murine GM-CSF for 6–8 days to harvest BMDM or BMDC, respectively34 (link),35 (link). For IFN-α treatment, human primary plasmacytoid dendritic cells (pDC) and myeloid dendritic cells (mDC) were isolated35 (link),58 (link) and treated with IFN-α (100 U/ml) for 2 h. Mouse splenic CD11c + DCs were isolated from spleen of WT and PARP9 KO mice without or with VSV infection for 1 day using anti-CD11c microbeads (Miltenyi Biotec)34 (link).
3
Ago Protein Immunoprecipitation and Analysis
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THP-1 cells (TIB-202TM) were from ATCC. RPMI 1640 media and Fetal bovine serum (FBS) were purchased from Gibco. HEPES, penicillin/streptomycin, L-glutamine, Hanks’ balanced salt solution, M199 medium, folic acid, hemin, adenosine, Pepstatin A, Phorbol 12-myristate 13-acetate (PMA), PreScission enzyme (GE27-0843-01), and Amicon® Ultra-2 centrifugal filter devices (UFC200324) were purchased from Millipore Sigma. Anti-Ago1 (5053), anti-Ago2 (2897), anti-Ago3 (5054), anti-Lamin A/C (2032), anti-Calreticulin (12238), and anti-Calnexin (2679) were purchased from Cell Signaling. Anti-GST (SC-138), anti-HSC70 (HSP70) (SC-7298), and anti-Actin (SC-47778) were from Santa Cruz biotechnology. Anti-GAPDH (G041) was from Abm, and anti TRBP (Ab42018) was purchased from Abcam. RPMI 1640 media lacking L-glutamine, L-arginine, and L-lysine was purchased from Caisson labs. North2South Hybridization buffer (37549), North2South Hybridization Stringency Wash Buffer (37555), and Chemiluminescent Nucleic Acid Detection Module kit (89880) were purchased from ThermoScientific.
4
THP-1 Monocyte Differentiation and Response
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THP-1 human monocytes (ATCC® TIB-202TM) were maintained in THP-1 complete medium (THP-1 CCM) and cultured in a controlled atmosphere at 37 °C. Macrophage differentiation was induced by incubation with phorbol myristate acetate (PMA) for 24 h. The culture medium was then replaced, and the cells were left with fresh medium for further 24 h before being exposed to the three functional ingredients, their mixture, and the inflammatory stimulus E. coli.
5
Cultivation of A549 and THP-1 Cell Lines
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The human alveolar epithelial cell line A549 (CCL-185TM, ATCC) was grown in DMEM/F12 culture medium containing 2 mM L-glutamine, 100 UI/mL penicillin, 100 µg/mL streptomycin, 5 mM Hepes and 10% fetal bovine serum (FBS). The human monocytes cell line THP-1 (TIB-202TM, ATCC) was grown in RPMI-1640 culture medium containing 2 mM L-glutamine, 0.05 mM 2-mercaptoethanol, 100 UI/mL penicillin, 100 μg/mL streptomycin, and 10% heat inactivated FBS (all reagents from GIBCO, Thermo Scientific, Illkirch-Graffenstaden, France). The two cell lines were grown in culture flasks at 37 °C in a 5% CO2 humidified chamber. For the experiments, the RPMI-1640 was used as culture medium in mono-cultures (A549 and THP-1) and co-cultures, after checking that medium change from DMEM-F12 (culture) to RPMI-1640 (experiment) did not impact the A549 cell growth. Co-cultures based on A549 and THP-1 cells have already been established in RPMI medium in the literature (e.g., Dekali et al. [21 (link)]).
6
Cultivation of Monocytic Cell Lines
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The U937–3xκB-LUC cells (kind gift from Prof Rune Blom-hoff),32 (link) a human monocytic cell line that was stably transfected with a luciferase reporter containing 3× NF-κB binding sites, were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 2 mM L- glutamine, 50 U/mL penicillin, 50 mg/mL streptomycin, and 75 μg/mL hygromycin. The human monocytic leukemia cell line THP-1 (purchased commercially; TIB-202TM; ATCC) was grown in suspension in complete RPMI 1640 culture medium supplemented with 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, 2 mM L-glutamine, and 0.05 mM β-mercaptoethanol. All solutions were obtained from Invitrogen (Carlsbad, CA, USA). Both cell lines were routinely maintained at 37°C in a humidified atmosphere of 5% CO2 and sub-cultivated 3 times per week. Because the properties of the cells can change dramatically after prolonged periods in culture, both the THP-1 and U937–3xκB-LUC cells were replaced by early frozen stock after 25 and 20 passages, respectively.
7
Cell Culture and Labeling Protocols
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The immortalized human monocyte cell line THP-1 (ATCC® TIB-202TM, VA, Gaithersburg, MD, USA) was used for cell labeling (passage of <20). The cells were maintained in culture as described previously [31 (link)].
The human adenocarcinoma cell line (MDA-MB-231, passage 46, ATCC® HTB-26TM, Gaithersburg, MD, USA) was cultured under the same conditions.
The human adenocarcinoma cell line (MDA-MB-231, passage 46, ATCC® HTB-26TM, Gaithersburg, MD, USA) was cultured under the same conditions.
8
Characterization of IL-37b Variants
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IL-37b WT, V1, V2, V1V2 were cloned into EF.CMV.RFP (addgene Plasmid #17619) downstream of the EF1a promoter and right clone were selected by sequencing. Constructed plasmids were transfected into HEK293T cells (ATCC CRL-3216TM) using Lipofectamine 2000 (Invitrogen). RFP positive cells were observed under fluoresce microscope. After 24 h transfection, cell lysate were collected for Western Blot analysis. IL-37b WT and V1 EF.CMV.RFP plasmids were also co-transfected with Caspase-1 plasmid into HEK293T cells; culture supernatant and cell lysate were collected for testing the interference of Variants to Casepase-1 cleavage of IL-37 by ELISA and Western Blot. IL-37b WT and V1 EF.CMV.RFP plasmids were also co-cultured with THP-1 (ATCC TIB-202TM) cells for 24 h, then 50 ng/ml LPS were added into co-culture system to stimulate THP-1 cells purchased from ATCC for 8 h. Culture supernatant were collected to detect IL-1β level by ELISA (DY201, R&D system).
9
Immune Response Assessment of Hydrogels
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To assess an immune response of the hydrogels, we prepared three different groups in 24-well culture plates (n = 4, each group): tissue culture plate (TCP), PH hydrogel-coated TCP, and PH/sFDM hydrogel-coated TCP. The coating volume of each hydrogel was 100 µL per well and the hydrogel-coated plates were kept at 37 °C for 3 min to induce gelation. Human peripheral blood monocytes (THP-1, TIB®-202TM; ATCC, Manassas, VA, USA) were seeded in the prepared culture plates (TCP, PH, and PH/sFDM) at the density of 3 × 105 cell/mL and cultivated for 3 days with Roswell Park Memorial Institute (RPMI) media (Gibco, Waltham, MA, USA), supplemented with 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin under normal culture condition. To measure the amount of VEGF released from THP-1 grown on different substrates, each culture medium was centrifuged and the supernatant was collected, followed by the measurement of VEGF concentration using VEGF ELISA kit (DVE00; R&D system).
10
THP-1 Macrophage Co-Culture Protocol
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