Computational protocol: The NLRP3 and NLRP1 inflammasomes are activated in Alzheimer’s disease

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Protocol publication

[…] Unstimulated, LPS- or Aβ42- primed, or LPS-primed and Aβ42-stimulated-PBMC were cultured on chamber slide (Lab Tek Nalge Nunc Intern. Naperville IL USA) for 24 h at 37 °C. Non-adherent cells were then washed away and monocytes were grown on chamber slides, fixed in 4 % paraformaldehyde in PBS for 15 min, and treated for 1 h at room temperature with FLICA staining of active caspase 1 or caspase 8 using green fluorescent FAM-YVAD-FMK probes, following the procedures suggested by the manufacturer (AM-FLICA). Cells were then treated with FIX and PERM Cell kit (eBioscience), and stained with PE or APC or FITC conjugated -mAbs specific for NLRP3 or NLRP1 and PYCARD or caspase 5 for 24 h at 4 °C. Finally cells were fixed with paraformaldehyde 1 % for 15 min, washed and mounted on slides using the Vectashield Mounting Medium (Vector Laboratories, Inc, Burlingame, CA, USA). Fluorescent images were acquired on a Leica TCS DMRE spectral laser-scanning confocal microscope (Leica Microsystems, Wetzlar, Germany) with the appropriate filters and laser (488, 633) and a 63× objective lens. Image analysis was performed using the Leica Confocal Software and co-localization index with ImageJ Software. Co-localization indexes were calculated using the plug in JACoP (Justo Another Co-localization Plugin) []. The summarized co-localization efficiency data was expressed as Pearson correlation coefficient (PCC) as previously described [–]. Briefly this coefficient measures the significance of true co-localization. The significance test evaluates the probability that the measured value of r obtained from the two colours is significantly greater than values of r that would be calculated if there were only random overlap. The test is performed by randomly scrambling the blocks of pixels (instead of individual pixels, because each pixel’s intensity is correlated with its neighbouring pixels) in one image, and then measuring the correlation of this image with the other (unscrambled) image (Costes et al.) The test produces values in the range −1 + 1, 0 indicating that there is no discernible correlation and −1 and +1 meaning strong negative and positive correlations, respectively. […]

Pipeline specifications

Software tools ImageJ, JACoP
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Homo sapiens
Diseases Alzheimer Disease