THP-1-dual reporter cells (InvivoGen) were stably integrated with two inducible reporter constructs, allowing the activation of the NF-kB or IRF pathways to be detected via measurement of secreted alkaline phosphatase or luciferase activity, respectively. At different time points post poly (dA:dT), poly I:C (4 μg/mL), or virus stimulation, 20 μL aliquots of media were sampled into 96-well plates. One hundred μL of Quanti-Luc luciferase substrate (InvivoGen) were added to each well, and plates were read immediately for luciferase activity using a GloMax 96 Microplate Luminometer (Promega).
Thp 1 dual reporter cells
Manufactured by InvivoGen
THP-1-dual reporter cells are an in vitro cell line derived from human monocytic leukemia cells. They are engineered to express two reporter genes that can be used to monitor the activation of specific signaling pathways.
Automatically generated - may contain errors
Lab products found in correlation
Quanti luc luciferase substrate, by InvivoGen (2 mentions)
Glomax 96 microplate luminometer, by Promega (2 mentions)
Thp 1 cells, by Japanese Collection of Research Bioresources Cell Bank (1 mentions)
Vero tmprss2 cells, by Japanese Collection of Research Bioresources Cell Bank (1 mentions)
Hct 8 cells, by American Type Culture Collection (1 mentions)
Calu 3 cells, by American Type Culture Collection (1 mentions)
Mrc 5 cells, by American Type Culture Collection (1 mentions)
293t cells, by RIKEN BioResource Center (1 mentions)
Vero cells, by RIKEN BioResource Center (1 mentions)
Penicillin, by InvivoGen (1 mentions)
Streptomycin, by InvivoGen (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by InvivoGen (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Zeocin, by InvivoGen (1 mentions)
Blasticidin, by InvivoGen (1 mentions)
4 protocols using thp 1 dual reporter cells
1
Monitoring NF-κB and IRF Pathway Activation
2
Cell Line Maintenance for Virus Research
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293T cells (human embryonic kidney cell line) and Vero cells (African green monkey kidney cell line) were obtained from the RIKEN BioResource Center Cell Bank. Calu-3 cells (human bronchial epithelial cell line), MRC5 cells (human fetal lung fibroblast cell line) and HCT8 cells (human rectal adenocarcinoma cell line) were obtained from the American Type Culture Collection (ATCC). THP-1 cells and Vero/TMPRSS2 cells53 (link) were obtained from the Japanese Collection of Research Bioresources Cell Bank. 293T, Calu-3 and MRC5 cells were maintained in Dulbecco’s modified Eagle’s Medium (DMEM) containing 10% fetal calf serum (FCS). HCT8 and THP-1 cells were maintained in RPMI-1640 medium with 10% FCS. Vero cells and Vero/TMPRSS2 cells were maintained in DMEM containing 10% FCS. 1 mg/mL G418 (Invivogen) was added to the growth medium for Vero/TMPRSS2 cells. THP-1-dual reporter cells, THP-1-dual KO-RIG-I cells and THP-1-dual KO-MDA5 cells were purchased from InvivoGen and maintained according to the manufacturer’s instructions.
3
Monitoring NF-κB and IRF Pathway Activation
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THP-1-dual reporter cells (InvivoGen) were stably integrated with two inducible reporter constructs, allowing the activation of the NF-kB or IRF pathways to be detected via measurement of secreted alkaline phosphatase or luciferase activity, respectively. At different time points post poly (dA:dT), poly I:C (4 μg/mL), or virus stimulation, 20 μL aliquots of media were sampled into 96-well plates. One hundred μL of Quanti-Luc luciferase substrate (InvivoGen) were added to each well, and plates were read immediately for luciferase activity using a GloMax 96 Microplate Luminometer (Promega).
4
Characterization of TLR10 in THP-1 Cells
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THP-1 (ATCC TIB-202) cells were obtained from the ATCC and cultured in RPMI-1640 (Life Technologies) supplemented with 10% fetal bovine serum (FBS, Life Technologies), 100 U/ml penicillin and 100 µg/ml streptomycin (Life Technologies). The TLR10 KD and TLR10 overexpressed (OE) THP-1 cells were generated and maintained as described previously (21 (link)). KD and overexpression of TLR10 was verified by RT-qPCR. THP-1-dual reporter cells were obtained from InvivoGen and maintained in RPMI-1640 culture medium with 10% FBS supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 10 µg/ml blasticidin (InvivoGen), and 100 µg/ml Zeocin (InvivoGen). Human peripheral blood monocytes were isolated from blood packs of healthy donors provided by the Hong Kong Red Cross Blood Transfusion Service and purified by adherence and differentiated into macrophages as described (21 (link)). Consent from blood donors was obtained by Hong Kong Red Cross to use blood components for research experiments. The work involved the use of human blood samples has been reviewed and obtained human ethics approval (ref no. UW 10-201, UW 14-170) issued by Institutional Review Board of the University of Hong Kong and met the standards of the Declaration of Helsinki.
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