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Inhibitor Mechanism |
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Endosomal toll-like receptor inhibitor (antagonist); Inhibitor of endosomal acidification on which functional activity of endosomal TLRs (particularly TLR9 and TLR3) is dependent. |
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Background |
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Chloroquine is a weak base which can partition into acidic vesicles such as endosomes and lysosomes, resulting in inhibition of endosomal acidification and lysosomal enzyme activity. Because acidic pH of endosomes is a prerequisite of endosomal TLR activation, chloroquine can serve as an antagonist for endosomal TLRs. Chloroquine and its analog quinacrine are also known to act as therapeutic agents for autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus, of which therapeutic activity is due to suppression of TLR9 activity as shown by researchers. |
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Solubility |
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Deionized water |
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Usage |
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Product Handling Protocol:
1. To make 100 mM stock solution, dissolve 200 mg chloroquine in 3.9 ml water by gentle vortex.
2. Filter sterilize through a 0.22 µm filter.
3. Store at 4oC (Note: Chloroquine solution is light sensitive).
4. For TLR signaling inhibition study, perform a pilot inhibitory test with the different concentrations of chloroquine ranging from 1 to 10 mM to optimize your experiments.
Note: See our validation tests using the IML-109 (chloroquine sensitive) and IML-102 (chloroquine insensitive) cell lines as shown in Figures 1 and 2. |
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Figure 1. Evaluation of inhibitory activity of chloroquine. TLR9/NF-kB/SEAPorter™ HEK 293 (IML-109) and TLR2/NF-kB/SEAPorter™ HEK 293 (IML-102) cells were plated in 96-well plates at 5x10^4 cells/well for 16 h. Cells were preincubated with different concentrations of chloroquine (0.05, 0.1, 0.5, 1, 5 and 10 µM) for 30 min. Cell were then stimulated with 10 µg/ml CpG (IMG-2209Hpt) [A] or 20 ng/ml MALP-2 (IMG-2206) [B] for 24 h. Secreted alkaline phosphatase (SEAP) was analyzed using SEAPorter™ Assay Kit (10055K). |
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Figure 2. IC50 evaluation of chloroquine. TLR9/NF-kB/SEAPorter™ HEK 293 (IML-109) cells were plated in 96-well plates at 5x10^4 cells/well for 16 h. Cells were preincubated with various concentrations of chloroquine for 30 min, followed by stimulation with 10 µg/ml CpG (IMG-2209Hpt) for 24 h. SEAP was analyzed using SEAPorter™ Assay Kit (10055K). Dose-responsive percent inhibition of each sample well was calculated to yield the chloroquine IC50 value.
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Reference |
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1. Macfarlane, D. E. et al. (1998). Antagonism of immunostimulatory CpG-oligodeoxynucleotides by quinacrine, chloroquine, and structurally related compounds. J. Immunol. 160, 1122-1131.
2. Rutz, M. et al. (2004). Toll-like receptor 9 binds single-stranded CpG-DNA in a sequence- and pH-dependent manner. Eur. J. Immunol. 34, 2541-2550.
3. Marshak-Rothstein, A. (2006). Toll-like receptors in systemic autoimmune disease. Nat. Rev. Immunol. 6, 823-835.
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Research purposes only.
Not for diagnostic or in vivo use.
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