All ELISAs with the exception of the hIL-18 and mIL-18 ELISA (MBL International antibodies D044-3, D045-6 and Thermofisher #BMS267-2, respectively) were from R&D Systems (#DY210, DY201, DY401, DY400, DY200). Quantification of cell death was performed by analysis of LDH release in the cell supernatant. Cell death was also quantified by monitoring propidium iodide (PI) incorporation. Briefly, 3 to 5 × 104 U937 cells, hMDMs or BMDMs were seeded in 0.3 cm2 wells of a black 96-flat-bottom-well plate. One hour after infection, PI was added at a final concentration of 5 μg.mL−1. Fluorescence was measured over a 24 h period on a microplate fluorimeter (Tecan).
Microplate fluorimeter
Manufactured by Tecan
Sourced in France
The Microplate fluorimeter is a lab equipment designed to measure the fluorescence intensity of samples in a microplate format. It is used to quantify fluorescent molecules or compounds in a high-throughput manner.
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Lab products found in correlation
4 protocols using microplate fluorimeter
1
Quantification of Cell Death in Immune Cells
2
Bioluminescent P. aeruginosa PAKlumi Protocol
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We used the bioluminescent P. aeruginosa strain PAKlumi that constitutively expresses the luxCDABE operon from Photorhabdus luminescens46 (link). PAKlumi was grown in Lennox Broth (LB), at 37°C with shaking at 180 rpm.
Phage PAK_P1 lysates were propagated on PAKlumi as previously described 47 (link). PAK-P1 was added to mid-log culture of PAKlumi at a multiplicity of infection (MOI) of 0.001. The infected culture was kept at 37°C with shaking until lysis occured. The culture was centrifuged (7000 rpm, 10 min, 4°C) and the supernatant filtered (0.2 μm) and then concentrated in Phosphate Buffered Saline (PBS) (Sigma) by ultrafiltration (Vivaflow 200™; Sartorius). The lysates were purified by cesium chloride density gradients, followed by dialysis in water and PBS. Endotoxins were removed by passages through an EndoTrap HD™ column (Hyglos, Germany) three times. Endotoxin levels were measured using the Endonext™ kit (Biomerieux) and a microplate fluorimeter (Tecan). The endotoxin level measured for the lysate was 10.05 EU/mL.
Bacteria from lung homogenates were enumerated by plating on cetrimide + 1 % glycerol agar plates (Sigma-Aldrich) and incubated overnight in 37°C. Phage was enumerated by spotting serial dilutions on LB agar plates that were previously inundated by a fresh culture of PAKlumi and dried under a safety cabinet 30 min. before use.
Phage PAK_P1 lysates were propagated on PAKlumi as previously described 47 (link). PAK-P1 was added to mid-log culture of PAKlumi at a multiplicity of infection (MOI) of 0.001. The infected culture was kept at 37°C with shaking until lysis occured. The culture was centrifuged (7000 rpm, 10 min, 4°C) and the supernatant filtered (0.2 μm) and then concentrated in Phosphate Buffered Saline (PBS) (Sigma) by ultrafiltration (Vivaflow 200™; Sartorius). The lysates were purified by cesium chloride density gradients, followed by dialysis in water and PBS. Endotoxins were removed by passages through an EndoTrap HD™ column (Hyglos, Germany) three times. Endotoxin levels were measured using the Endonext™ kit (Biomerieux) and a microplate fluorimeter (Tecan). The endotoxin level measured for the lysate was 10.05 EU/mL.
Bacteria from lung homogenates were enumerated by plating on cetrimide + 1 % glycerol agar plates (Sigma-Aldrich) and incubated overnight in 37°C. Phage was enumerated by spotting serial dilutions on LB agar plates that were previously inundated by a fresh culture of PAKlumi and dried under a safety cabinet 30 min. before use.
3
Cell Lysis and Enzyme Assay Protocol
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After 24 h of treatment with RSV or suramin, cells were collected and disrupted with a RIPA lysis buffer containing a protease inhibitor mix. 10-μl samples were mixed with 40 μl assay buffer, followed by incubation for 30 min at 37 °C, addition of 5 μl developing solution and incubation for an additional 10 min at 37 °C. The fluorescence signal was detected using a microplate fluorimeter (Tecan, Hillsborough, NC).
4
Quantifying Toxin Cytotoxicity on Cells
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Toxin cytotoxicity to osteoclasts and macrophages was quantified by monitoring propidium iodide (PI) incorporation as previously described [13 (link)]. Cells were incubated with PI (5 μg/ml) and variable concentrations of recombinant staphylococcal toxins. Propidium iodide fluorescence was measured over a 3-hour period on a microplate fluorimeter (Tecan, Lyon, France), using untreated cells as control.
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