Whole blood cell cultures were performed to determine the intracellular levels of cytokines. Briefly, whole blood was diluted 1:1 with RPMI-1640 medium, supplemented with penicillin/streptomycin (100 U/100 mg/ml), L-glutamine (2 mM), and HEPES (10 mM) (all from Invitrogen) and distributed in 12-well tissue culture plates (Costar). The cultures were then stimulated with PPD, ESAT-6, CFP-10 or anti-CD3 or media alone in the presence of the costimulatory molecules, CD49d /CD28 at 37° C for 6 hrs. Brefeldin A (10 μg/ml) was added after 2 hours. After 6 hours, centrifugation, washing and red blood cell lysis was performed. The cells were fixed using cytofix/cytoperm buffer (BD Biosciences) and cryopreserved at −80°C. For neutralization experiments, whole blood was cultured in the presence of anti-IL-1R (5 μg/ml), anti-IL-6R (5 μg/ml) and anti TNFR1 (5 μg/ml) (R& D Systems) or isotype control antibody (5 μg/ml) (R&D Systems) at 37°C for 1 h following which PPD was added and cultured for an additional 23 h.
Anti il 6r
Manufactured by R&D Systems
Anti-IL-6R is a recombinant protein used in cell culture and biochemical assays to study the functions of the interleukin-6 receptor (IL-6R). It binds to and blocks the activity of IL-6R, which is involved in regulating various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
12 well tissue culture plate, by Corning (1 mentions)
Isotype control antibody, by R&D Systems (1 mentions)
Hepes, by Thermo Fisher Scientific (1 mentions)
Anti tnfr1, by R&D Systems (1 mentions)
Cytofix cytoperm buffer, by BD (1 mentions)
Tgf β, by R&D Systems (1 mentions)
Lsrfortessa, by BD (1 mentions)
Bovine albumin, by Sartorius (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Recombinant human il 6, by R&D Systems (1 mentions)
Specific sirna, by Thermo Fisher Scientific (1 mentions)
Ht 29, by American Type Culture Collection (1 mentions)
Ccd 18co, by American Type Culture Collection (1 mentions)
Cryptotanshinone, by Selleck Chemicals (1 mentions)
Stattic, by Selleck Chemicals (1 mentions)
Ly294002, by Selleck Chemicals (1 mentions)
U0126, by Selleck Chemicals (1 mentions)
Anti il 6, by R&D Systems (1 mentions)
5 protocols using anti il 6r
1
Intracellular Cytokine Profiling in Whole Blood
2
Modeling Alveolar Epithelial Cell Response
3
Multiparametric Flow Cytometry of Liver NKCs
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The trNK cells isolated from harvested mice livers were diluted to a concentration of 1 million cells per milliliter in a saline buffer supplemented with 1% bovine albumin (Biological Industries; Cat# 02–023-5A). Subsequently, the cells were labeled with the following antibodies. Anti-mouse NK1.1 (murine NK cell marker) (Biogems; Cat# 83712–70), anti-CD49a (MACS; Lot# 5150716246), anti CD49b (MACS; Lot# 5150716256), anti-mouse lysosomal-associated membrane protein-1 (CD107a; NK1.1 cells cytotoxicity marker, eBioscience, Cat# 48–1071), and anti-IL-6 R (R&D; Cat# 48–1044) were used. All antibodies were incubated for 40 min at 4°C. pHSCs (106 cells/mL) were stained with rabbit anti-mice αSMA (R&D; IC1420P). The cells were washed with 0.5 mL staining buffer and fixed with 20 mL 2% paraformaldehyde. All stained cells were analyzed with a flow cytometer (BD LSR Fortessa™, Becton Dickinson, Immunofluorimetry systems, Mountain View, CA).
4
Colon Cancer Cell Lines and Inhibitors
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CRC cell lines HT-29 and Colo205 and fibroblast cell line CCD-18Co from the colon were purchased from ATCC. Human normal colon mucosal epithelial cell line NCM460 was obtained from INCELL. HT-29 and Colo205 were cultured in complete Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), with essential supplementation including penicillin/streptomycin solution, while NCM460 in complete RPMI-1640 medium. CCD-18Co was cultured in ATCC-formulated Eagle's Minimum Essential Medium (#30-2003) with 10% FBS. All the cells were incubated at 37°C in a 5% CO2 humidity atmosphere. The recombinant human IL-6 was purchased from R&D Systems. For the function blocking, the specific antibody 10D5 against integrin β6 was purchased from Merck Millipore and neutralizing anti-IL-6 as well as anti-IL-6R from R&D Systems. To knock down integrin β6 (#144659), IL-6R (#106147), or STAT-3 (#116558), cells were transfected with specific siRNA (Thermo Fisher Scientific) using Lipofectamine 3000 (Invitrogen) according to the manufacturer's instructions. For the inhibition of the STAT-3 pathway, specific STAT-3 inhibitors Stattic and Cryptotanshinone (CTS), ERK/MAPK inhibitor U0126, and PI3K inhibitor LY294002 were purchased from SelleckChem.
5
Modulation of RA-FLS Proliferation
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HEK293T cells were transfected with mc_mock, mc_sTNFR-Fc, or mc_anti-IL-6R and the conditioned media were collected 24 h post-transfection. RA-FLS were incubated with or without human IL-6 (100 ng/mL; R&D systems, Minneapolis, MN), sIL-6R (100 ng/mL; PeproTech, Rocky Hill, NJ), and TNFα (20 ng/mL; R&D systems) for 72 h. During the incubation, RA-FLS were treated with the conditioned media of HEK293T cells transfected with mc_mock, mc_sTNFR-Fc, mc_anti-IL-6R or PBS (as a negative control). Cell proliferation was assessed using the cell counting kit-8 (CCK-8; Dojindo Molecular Technologies, Rockville, MD), according to the manufacturer's instructions.
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