U2OS cells (WT or G3BP1/2 KO cells) were seeded with a Thermo MultiDrop dispenser into a Greiner μClear 384 well plate (Greiner Cat #781092) and treated as described in the previous sections. Cells were fixed with 3.7% formaldehyde diluted in PBS for 10 min, washed with PBS and subsequently permeabilized with 0.2% Triton X-100 in PBS for 10 min. Cells were then blocked with 3% BSA in PBS for 1 hr and incubated overnight with primary antibodies (goat anti-eIF3η (Santa Cruz Biotechnology, sc-16377) diluted 1:2000 and rabbit anti-G3BP1 (Thermo Fisher, PA5-29455) diluted 1:500 in blocking solution). After washing with PBS, cells were incubated for 1 h with secondary antibodies (donkey anti-goat Alexa-647 and donkey anti-rabbit Alexa 488 diluted 1:1000 in blocking solution). The cytoplasm was stained with CellMaskBlue (Invitrogen) and the nuclei were stained with Hoechst (Invitrogen). Images were acquired on an automated confocal microscope Yokogawa cv7000 with a 40x 0.95 NA air lens.
Multidrop dispenser
Manufactured by Greiner
The MultiDrop dispenser is a versatile lab equipment designed for precise liquid dispensing. It features a compact and robust construction, enabling accurate and reproducible liquid transfers across a range of applications.
Automatically generated - may contain errors
Lab products found in correlation
Cellmask blue, by Thermo Fisher Scientific (1 mentions)
Rabbit anti g3bp1, by Thermo Fisher Scientific (1 mentions)
μclear 384 well plate, by Greiner (1 mentions)
Goat anti eif3η, by Santa Cruz Biotechnology (1 mentions)
Hoechst, by Thermo Fisher Scientific (1 mentions)
Celltiter glo, by Promega (1 mentions)
2 protocols using multidrop dispenser
1
Subcellular Localization of eIF3η and G3BP1
2
High-throughput Cytotoxicity Assay Protocol
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Cells were assayed in growth media at a density of 500–1,000 cells/well. Five μL of cells were dispensed into 1,536-well, white, solid-bottom, TC-treated plates (Greiner Bio One) using a Multidrop dispenser and incubated at 37°C, 5% CO2, under a humidified atmosphere for 5 hours. Twenty three nL of compounds and controls (neutral control DMSO or positive control bortezomib at final concentration of 2.3 μM) were subsequently transferred by Kalypsys pintool. For primary screens, the MIPE collection was tested at 11-point dilutions (final concentration range from 0.78 nM to 46 μM) and the NPC collection at 8-point dilutions (final concentration range from 0.59 nM to 46 μM). Compounds tested in follow-up screens were assayed at 11-point dilutions (final concentration range from 0.78 nM to 46 μM). Cells were incubated for 48 hours, followed by addition of 3 μL of CellTiter-Glo (Promega), then after a ∼15 minute incubation at RT, samples were analyzed for luminescence intensity using a ViewLux High-throughput CCD imager equipped with clear filters.
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