For experiments, colonoids were passaged using TrypLE techniques as already described but with modifications3 (link), 27 (link), 29 (link). First, Matrigel domes were disrupted by adding ice-cold PBS containing 5 mM EDTA and incubating on ice for 15 min. Colonoids were washed twice with base media, resuspended in TrypLE express (Gibco) and incubated at 37 °C with 5% CO2 for 2 × 5 min. The colonoids were then rapidly disrupted into single cell suspensions with repeated pipetting through a p1000 tip, and an equal volume of organoid media was added. Cells were centrifuged, then resuspended in base media. The cells were counted using an automated cell counter and the appropriate number of cells (15,000 cells/well for human; 10,000 cells/well for mouse) were seeded using Matrigel as described in the colonoid isolation section in a 24-well plate. Colonoids were stimulated (days 5–7 after seeding for mice, day 10–14 for human) with base media with FliC (100 ng/ml; InvivoGen) or human IL-1β (10 ng/mL; Sigma) or murine IL-1β (10 ng/mL; PeproTech) or LPS O55:B5 (1000 ng/ml; InvivoGen) with or without recombinant IL-37 (100 to 0,1 ng/ml; R&D) or corresponding volume of base media for 4 h.
Human il 1β
Manufactured by Merck Group
Sourced in United States
Human IL-1β is a recombinant protein that represents the human interleukin-1 beta cytokine. It is a pro-inflammatory mediator involved in the regulation of immune and inflammatory responses.
Automatically generated - may contain errors
Lab products found in correlation
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Murine il 1β, by Thermo Fisher Scientific (1 mentions)
Recombinant il 37, by R&D Systems (1 mentions)
Tryple express, by Thermo Fisher Scientific (1 mentions)
Lps o55 b5, by InvivoGen (1 mentions)
T0157, by Merck Group (1 mentions)
Kc7f2, by Selleck Chemicals (1 mentions)
Human tnf α, by Merck Group (1 mentions)
Rat tnf α, by Merck Group (1 mentions)
I3275, by Merck Group (1 mentions)
S7946, by Selleck Chemicals (1 mentions)
Human ifn γ, by Merck Group (1 mentions)
Alizarin red dye, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Penicillin and streptomycin, by Thermo Fisher Scientific (1 mentions)
Calcein am, by BioLegend (1 mentions)
Anti lc3b, by Abcam (1 mentions)
Collagen 1, by Merck Group (1 mentions)
Anti atg7, by Cell Signaling Technology (1 mentions)
Anti beclin1, by Abcam (1 mentions)
8 protocols using human il 1β
1
Passaging and Stimulation of Colonoids
2
Cytokine and HIF-1α Inhibitor Study
3
Osteogenic Differentiation of HEK-293 Cells
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Human Embryonic Kidney 293 cell line (HEK- 293) was purchased from the cell bank of Royan Institute for Stem Cell Biology and Technology (RI-SCBT). We used fetal bovine serum (FBS), penicillin and streptomycin and osteogenic differentiation medium Gibco (Gibco Life Technologies, Germany). Anti-LC3B, anti-Beclin 1, Anti-CD63, Anti- CD9, anti-CD81 antibodies, Anti- Calnexin antibodies, anti-beta actin-loading control antibodies, goat anti-rabbit IgG H&L (HRP) and anti-beta actin-loading control antibodies were purchased from Abcam, USA.
In addition, we used BCA Protein Quantification kit (Novagen, Iran), Calcein-AM (Bio legend, USA), and MTT solution (Life Technologies, England), difluoride (PVDF) membrane (Bio-Rad Laboratories, CA, USA) PBS Tween (Thermo Fisher Scientific, Massachusetts, USA), Minimum Essential Medium (MEM, Thermo Fisher Scientific, Massachusetts, USA) and Anti- Atg7 (cell signal, USA). We purchased Human IL-1β, collagen I, Human IL-4, Human IL10, TNF-α, TRI Reagent alkaline phosphatase, alizarin red dye, oil red dye, Curcumin, H2O2, BSA, PBS, glutaraldehyde, and 5% BSA from Sigma (Sigma Aldrich, USA).
In addition, we used BCA Protein Quantification kit (Novagen, Iran), Calcein-AM (Bio legend, USA), and MTT solution (Life Technologies, England), difluoride (PVDF) membrane (Bio-Rad Laboratories, CA, USA) PBS Tween (Thermo Fisher Scientific, Massachusetts, USA), Minimum Essential Medium (MEM, Thermo Fisher Scientific, Massachusetts, USA) and Anti- Atg7 (cell signal, USA). We purchased Human IL-1β, collagen I, Human IL-4, Human IL10, TNF-α, TRI Reagent alkaline phosphatase, alizarin red dye, oil red dye, Curcumin, H2O2, BSA, PBS, glutaraldehyde, and 5% BSA from Sigma (Sigma Aldrich, USA).
4
Cytokine Modulation in Immune and Skin Cells
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RAW 264.7 and HaCaT cells (5 × 106 cells/mL) were seeded in 6-well plates and cultured for 24 h. After treatment with CLE at different concentrations (25, 50, and 100 μg/mL) for 4 h, the cells were stimulated with LPS (200 ng/mL) or TNF-α/IFN-γ (10 ng/mL) for 24 h. DEX (5 µg/mL) was used as a positive control. Then, the culture supernatants were collected. The concentration of PGE2 in the supernatants of RAW 264.7 cells was detected using a PGE2 ELISA kit (Cat# E-EL-0034, Elabscience, Houston, TX, USA) according to the manufacturer’s instructions. HaCaT cells’ supernatants were analyzed using ELISA kits for TARC (EHCCL17, Invitrogen, Waltham, MA, USA), RANTES (EHRNTS, Invitrogen), Human IL-8 (BMS204-3, Invitrogen), Human Il-1β (RAB0273, Sigma Aldrich), and MCP-1 (BMS281, Invitrogen) according to the manufacturer’s instructions.
5
Inflammation Response in Normal and ALS Fibroblasts
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Normal and ALS fibroblast cells were seeded in 6-well plates and maintained until they reached 80% confluence before incubation in DMEM serum-free media (SFM). Fibroblast cells were washed five times with PBS and then treated with or without stimulation with 10 ng/mL LPS (Sigma, St. Louis, MO) for 3 h. To confirm the induction of inflammation, expression of human IL-1β was validated using RT-PCR. Expression of TOLLIP was validated by RT-PCR of the cultured cells after LPS treatment at passages 4, 8, and 12. Normal- and patient-derived iPSCs were washed five times with PBS and then treated with 1 μg/mL or 5 μg/mL LPS for 24 h, or remained without any treatment. Normal- and patient-derived iPSCs and neural rosettes were washed five times with PBS and then treated with 10 ng/mL or 100 ng/mL hIL-1β (R&D Systems, Minneapolis) for 3 h, or remained without any treatment. To confirm the induction of inflammation, expression of COX-2 was validated using RT-PCR. Expression of TOLLIP was validated by RT-PCR of the cultured cells after hIL-1β treatment.
6
IL1-β Induced Organoid Modulation
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Day-21 TSC2+/+ organoids were incubated in Advanced RPMI + 1X L-GlutaMAX containing 50 ng/ml human IL1-β (Sigma-Aldrich # H6291) for 96hs29 (link). The medium containing IL1-β was replaced every 48 h.
7
Curcumin SNEDDS and SMEDDS Anti-Inflammatory Effects
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To investigate the anti-inflammatory effect of the curcumin containing SNEDDS and SMEDDS, ELISA tests were performed on HaCaT and Caco-2 cell lines. Cells were seeded on 96-well plates at a density of 104 cells/well. When the cells fully grow over the wells’ membrane, culture media was removed. The cells were incubated with the samples for 1 h. Samples were prepared by dissolving curcumin containing extract (Cur-extr), SNEDDS and SMEDDS in PBS at different concentrations (2 w/w%, 5 w/w%, 10 w/w%). After samples were removed, 50 μL of IL-6 (30 ng/mL) was added to the cells and incubated overnight in order to induce inflammation. The next day the supernatant was removed, and human IL-1β (Sigma—RAB0273) and TNF-α ELISA Kits (Sigma—RAB0476) were used according to the manufacturer’s instructions.
8
IL1-β Stimulation of TSC2 Organoids
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(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted October 20, 2021. ; https://doi.org/10.1101/2021.10.20.465120 doi: bioRxiv preprint Day-21 TSC2 +/+ organoids were incubated in Advanced RPMI + 1X L-GlutaMAX with 50ng/ml human IL1-β (Sigma-Aldrich # H6291) for 96hs, as previously described (25) .
The medium containing IL1-β was replaced every 48h.
The copyright holder for this preprint this version posted October 20, 2021. ; https://doi.org/10.1101/2021.10.20.465120 doi: bioRxiv preprint Day-21 TSC2 +/+ organoids were incubated in Advanced RPMI + 1X L-GlutaMAX with 50ng/ml human IL1-β (Sigma-Aldrich # H6291) for 96hs, as previously described (25) .
The medium containing IL1-β was replaced every 48h.
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