To prepare medium with 6 mM Mg2+, equi-osmolar MgSO4 solution was prepared and added to RPMI and DMEM media (149 and 175 mOsM, respectively). Heat-inactivated FBS was prepared by incubation at 75 °C for 5 min in 1 mL aliquots. 3T3 and HEK-293 cells were grown in DMEM medium, and H441 cells were grown in RPMI medium. Cells were grown to confluency in appropriate medium supplemented with 10% freshly thawed FBS (Gibco). 3T3 (2.5 × 103 cells/mL), HEK-293 (1 × 104 cells/mL), and H441 (1.5 × 104 cells/mL) cells were seeded by adding 200 μL of cell suspension into the wells (n = 5) of a 96-well plate for each time point (0, 1, 3, 5, or 7 days after seeding), in modified medium prepared as follows: medium + 10% FBS, medium + 10% FBS + 6 mM Mg2+, medium + 10% heat-inactivated FBS, medium + 10% heat-inactivated FBS + 6 mM Mg2+, medium + 10% FBS + 200 nM actin, and medium + 10% FBS + 200 nM actin + 6 mM Mg2+. At each time point, cell proliferation was measured by CyQuant Direct Cell proliferation assay (Life Technologies) following the vendor protocol exactly. Fluorescence was measured on a BioTek NEO HTS plate reader with excitation at 480 nm and emission at 530 nm.
Neo hts plate reader
Manufactured by Agilent Technologies
The NEO HTS plate reader is a high-throughput spectrophotometric instrument designed for various analytical applications in life science research. It provides accurate and reliable measurements of absorbance, fluorescence, and luminescence signals in microplates.
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3 protocols using neo hts plate reader
1
Cell Proliferation in Mg2+ Supplemented Media
2
Optimizing SHERLOCK P. falciparum Detection in Whole Blood
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Using live intraerythrocytic P. falciparum spiked into whole blood as a simulated malaria sample, we trialed multiple sample preparation methods. All sample preparation methods tested had a final volume of 20 μL with a final P. falciparum concentration of 1 fM or 602 copies per microliter (various methods had different dilution steps and so initial spiked concentration varied) and were tested via rehydration of the one-pot lyophilized SHERLOCK P. falciparum pellet described below. Fluorescence was measured over 1 h at 40 °C using a BioTek NEO HTS plate reader with readings every 3 min (excitation: 485 nm; emission: 535 nm). Detergents at varying wt/vol% (SDS 0.5%, saponin 1%, Tween-20 1%, Triton-X 100 1%) were added to a 20-μL simulated whole-blood sample along with 100 mM TCEP. Two heating sample preparation protocols were tested: 1) dilution of simulated sample 1:4 in nuclease-free water followed by 10-min 95 °C incubation (1:4 dilution required to prevent solidification when diluting with water), and 2) addition of 100 mM TCEP into the diluted simulated sample prior to 10-min 95 °C incubation. For optimization of chemical deactivation methods of nucleases and SHERLOCK inhibitors, combinations of chelators and reducing agents added to 20-μL simulated samples at concentrations demonstrated in Fig. 3B were tested.
3
Cell Proliferation Assay with Nanostructure
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The same methods from the cell proliferation assay were used to modify medium. Each cell line was seeded by adding 100 μL of 105 cells/mL cell suspension into wells (n = 5) of a 96-well plate for control or experimental groups in the following medium conditions: medium + 10% FBS, medium + 10% heat-inactivated FBS + 6 mM Mg2+, and medium + 10% FBS + 200 nM actin + 6 mM Mg2+. Five microliters of 100 ng/μL DNO was added to the experimental group after overnight incubation to allow cell attachment. Twenty-four hours following addition of the nanostructure, cell viability was measured by CellTiter-Glo Luminescent cell viability assay (Promega) following the vendor protocol exactly. Luminescence signal was measured on a BioTek NEO HTS plate reader.
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