Protocols for Crystallizing and Transfecting Cells

ATP, Ultrapure LPS, colchicine, nocodazole, MDP, Phorbol 12-myristate 13-acetate (PMA), and doxycycline were obtained from Sigma. Imject Alum was obtained from Thermo. Nigericin, flagellin, poly (dA:dT) and vinblastine were obtained from Enzo. iE-DAP was obtained from Invivogen. MSU crystals and cholesterol crystals were made as described46 (link)47 (link): cholesterol (Sigma) dissolved in 95% ethanol (12.5 g l⁻¹) was heated to 60 °C, filtered through filter paper and left at room temperature to allow crystallization; 8 g of uric acid (Sigma) was dissolved in 1,600 ml of boiling water containing 49 ml NaOH, and then PH was adjusted to 7.2, and crystals were formed by gradual cooling down. DOTAP (Roche), TransIT/LT1 (MirusBio), and TransIT/TKO (MirusBio) were used as transfection reagents. Mitotracker Red, Mitotracker Deep Red FM, TubulinTracker Green, Hoechst 33342, DAPI were obtained from Invitrogen. CytoTox 96 non-radioactive cytotoxicity assay (Promega) was used to measure lactate dehydrogenase (LDH) as an indication of cell viability following manufacturer’s instruction.

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Li X., Thome S., Ma X., Amrute-Nayak M., Finigan A., Kitt L., Masters L., James J.R., Shi Y., Meng G, & Mallat Z. (2017). MARK4 regulates NLRP3 positioning and inflammasome activation through a microtubule-dependent mechanism. Nature Communications, 8, 15986.

Publication 2017

Alum Assay Cell viability Cholesterol Colchicine Crystallization Cytotox Dapi Dotap Doxycycline Ethanol Flagellin Hoechst 33342 Ie dap Lactate dehydrogenase Nigericin Nocodazole Phorbol myristate acetate Poly Promega Radioactive Transfection Uric acid Vinblastine

Corresponding Organization : Inserm

Other organizations : Shanghai Jiao Tong University, Shanghai Institute of Hematology, Medizinische Hochschule Hannover, The University of Texas Health Science Center at San Antonio

Top 5 similar protocols

Variable analysis

independent variables

Ultrapure LPS
Colchicine
Nocodazole
Phorbol 12-myristate 13-acetate (PMA)
Doxycycline
Imject Alum
Nigericin
Flagellin
Poly (dA:dT)
Vinblastine
IE-DAP
MSU crystals
Cholesterol crystals
DOTAP
TransIT/LT1
TransIT/TKO

dependent variables

Lactate dehydrogenase (LDH) as an indication of cell viability

control variables

Mitotracker Red
Mitotracker Deep Red FM
TubulinTracker Green
Hoechst 33342

positive controls

CytoTox 96 non-radioactive cytotoxicity assay (Promega)

negative controls

None specified

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