hCMEC/D3 or hDMEC supernatants were collected after 4, 8, 12, 24 or 48 h of ICAM-1 cross-linking. ELISA was performed using the LEGEND MAX Human TNFα ELISA Kit with Pre-coated Plates (Biolegend) accordingly to the manufacturer’s instructions. Multi-Analyte Flow assay of chemokines relevant to neuroinflammation (28 (link)) was performed using the LEGENDplex Multi-Analyte Flow Assay Kit (Biolegend). Optionally anti-TNFα antibodies (1μg/ml, R&D SYSTEMS, Abingdon, UK) were added throughout the ICAM-1 stimulation period.
Anti tnf α antibodies
Manufactured by R&D Systems
Sourced in United States
Anti-TNF-α antibodies are laboratory reagents that specifically bind and neutralize tumor necrosis factor alpha (TNF-α), a pro-inflammatory cytokine. These antibodies are used in research applications to study the role of TNF-α in various biological processes and disease models.
Automatically generated - may contain errors
3 protocols using anti tnf α antibodies
1
ICAM-1 Induced Neuroinflammation Profiling
2
HNSCC Cell Stimulation and CXCR4 Knockdown
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For cell line stimulation, CAL 27 were purchased from American Type Culture Collection (ATCC® CRL-2095) and the human HNSCC cell lines, HN 30, were obtained from the National Institutes of Health (Rockville, MD, USA). HN 30 cells were seeded at a density of 4 × 105 and cultured in DMEM supplemented with 10% FBS in a humidified atmosphere of 5% CO2, at 37 °C for 24 h. The cells were washed twice with 1× PBS and starved in 1% FBS-supplemented growth medium overnight. Next, cells were stimulated with different concentrations of pDC-CM, with or without anti-TNF-α antibodies or TNF-α (50 ng/mL; R&D Systems, USA) for 72 h. Experiments were repeated at a minimum of three times.
Lentiviraus-CXCR-4-shRNA was constructed at Gene Pharma (Shanghai, China). Cells were plated in 6‐well plates and infected with lentivirus for 24 h in a complete medium containing 5 μg/ml polybrene.
Lentiviraus-CXCR-4-shRNA was constructed at Gene Pharma (Shanghai, China). Cells were plated in 6‐well plates and infected with lentivirus for 24 h in a complete medium containing 5 μg/ml polybrene.
3
OSCC Cell Line Stimulation Protocol
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For cell line stimulation, cells were seeded at a density of 4 × 10 5 and cultured in DMEM supplemented with 10% FBS in a humidi ed atmosphere of 5% CO 2 , at 37 °C for 24 h. The cells were washed twice with 1× PBS and starved in 1% FBS-supplemented growth medium overnight. Next, cells were stimulated with different concentrations of pDC-CM, with or without anti-TNF-α antibodies or TNF-α (50 ng/mL; R&D Systems, USA) for 72 h. Experiments were repeated a minimum of three times.
The siRNA targeting CXCR-4 was purchased from Gene Pharma (Shanghai, China). The OSCC cell lines were cultured in 6-well cell culture plates and transfected with the siRNA using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer's protocol.
The siRNA targeting CXCR-4 was purchased from Gene Pharma (Shanghai, China). The OSCC cell lines were cultured in 6-well cell culture plates and transfected with the siRNA using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer's protocol.
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