Journal: Scientific Reports

Article Title: Time-lapse image analysis reveals trigger-dependent differences in ASC speck lifetime in the NLRP3 inflammasome

doi: 10.1038/s41598-026-50936-x

Figure Lengend Snippet: Parameter estimation. (A) ASC-GFP-specks in PMA-differentiated, LPS-primed THP-1 cells were counted both using FIJI (blue triangles) and the proposed algorithm (red circles) for a randomly selected field of view in one of the MSU-triggered wells. Algorithm parameters were selected to minimize the difference between the counts obtained from the two methods. (B-E) The estimated values were assessed by comparing the counts obtained from FIJI and the proposed algorithm for one randomly selected well triggered by either ATP or nigericin as well as for untreated controls and LPS-primed controls.

Article Snippet: Cells were primed with 500 ng/mL ultrapure LPS (Invivogen, San Diego, CA) for 4 hours, and triggering of inflammasome complex formation was performed by addition of either 5 mM ATP (Merck, Darmstadt, Germany), 100 μg/mL MSU (Invivogen, San Diego, CA) or 10 μM nigericin (Invivogen, San Diego, CA).

Techniques:

Journal: Scientific Reports

Article Title: Time-lapse image analysis reveals trigger-dependent differences in ASC speck lifetime in the NLRP3 inflammasome

doi: 10.1038/s41598-026-50936-x

Figure Lengend Snippet: Speck formation in THP-1 cells. Speck formation in PMA-differentiated, LPS-primed THP-1-ASC-GFP cells after triggering with either (A) ATP, (B) MSU or (C) nigericin. Specks (white arrows) are seen as bright, green spots. Figure shows representative images taken 1 h after addition of trigger; inserts are chosen to show ASC-GFP-speck formation. Transmission and GFP channels have been merged, with the GFP channel pseudo-colored green.

Article Snippet: Cells were primed with 500 ng/mL ultrapure LPS (Invivogen, San Diego, CA) for 4 hours, and triggering of inflammasome complex formation was performed by addition of either 5 mM ATP (Merck, Darmstadt, Germany), 100 μg/mL MSU (Invivogen, San Diego, CA) or 10 μM nigericin (Invivogen, San Diego, CA).

Techniques: Transmission Assay

Journal: Scientific Reports

Article Title: Time-lapse image analysis reveals trigger-dependent differences in ASC speck lifetime in the NLRP3 inflammasome

doi: 10.1038/s41598-026-50936-x

Figure Lengend Snippet: Speck detection in PMA-differentiated LPS-primed THP-1-ASC-GFP cells 2 h after the addition of (A) ATP, (B) MSU and (C) nigericin. ASC-GFP-specks are automatically identified and indexed and a bounding box is added. White arrows point to visually identifiable specks, which are detected successfully. Pink arrows point to the visually identifiable specks, which were not identified by the algorithm since they failed to meet the predefined intensity, size and/or circularity criteria. The figure shows representative images. Scale bars are 50 μm.

Article Snippet: Cells were primed with 500 ng/mL ultrapure LPS (Invivogen, San Diego, CA) for 4 hours, and triggering of inflammasome complex formation was performed by addition of either 5 mM ATP (Merck, Darmstadt, Germany), 100 μg/mL MSU (Invivogen, San Diego, CA) or 10 μM nigericin (Invivogen, San Diego, CA).

Techniques:

Journal: Scientific Reports

Article Title: Time-lapse image analysis reveals trigger-dependent differences in ASC speck lifetime in the NLRP3 inflammasome

doi: 10.1038/s41598-026-50936-x

Figure Lengend Snippet: Individual ASC-GFP-speck count. Mean number across the experiments of individual ASC-GFP-specks formed over 24 h in PMA-differentiated, LPS-primed THP-1-ASC-GFP cells after triggering speck formation with either ATP, MSU or nigericin. The mean was calculated using the sum of individual specks numbers from all fields of view for each biological replicate (see section Data analysis). ASC-GFP-specks were tracked over 24 h and quantified. Data were analyzed by one-way ANOVA with Tukey multiple comparisons test and are shown as mean ± SEM, n = 5. *p-adj < 0.05, n.s.: not significant.

Article Snippet: Cells were primed with 500 ng/mL ultrapure LPS (Invivogen, San Diego, CA) for 4 hours, and triggering of inflammasome complex formation was performed by addition of either 5 mM ATP (Merck, Darmstadt, Germany), 100 μg/mL MSU (Invivogen, San Diego, CA) or 10 μM nigericin (Invivogen, San Diego, CA).

Techniques:

Journal: Scientific Reports

Article Title: Time-lapse image analysis reveals trigger-dependent differences in ASC speck lifetime in the NLRP3 inflammasome

doi: 10.1038/s41598-026-50936-x

Figure Lengend Snippet: ASC-GFP-speck lifetime. Specks from PMA-differentiated, LPS-primed THP-1 cells were automatically identified and tracked after triggering with either ( A ) ATP, ( B ) MSU or ( C ) nigericin. ( D ) Survival curves of specks appearing throughout the experiment for different triggers. For each trigger, data from all fields of view in all replicates ( n = 5) were pooled and analyzed. The results were then truncated to eliminate the specks with a life cycle of 1 frame as artifacts. Dashed lines in (D) show median survival speck’s lifetime that is equal to 2.0, 4.5, and 4.0 h for ATP, MSU, and nigericin respectively.

Article Snippet: Cells were primed with 500 ng/mL ultrapure LPS (Invivogen, San Diego, CA) for 4 hours, and triggering of inflammasome complex formation was performed by addition of either 5 mM ATP (Merck, Darmstadt, Germany), 100 μg/mL MSU (Invivogen, San Diego, CA) or 10 μM nigericin (Invivogen, San Diego, CA).

Techniques:

Journal: Scientific Reports

Article Title: Time-lapse image analysis reveals trigger-dependent differences in ASC speck lifetime in the NLRP3 inflammasome

doi: 10.1038/s41598-026-50936-x

Figure Lengend Snippet: Time of formation and lifetime of ASC-GFP-specks. Relative number of specks formed at each timepoint and their duration, normalized to the total number of specks for each trigger ( n = 5). THP-1-ASC-GFP cells were PMA-differentiated, primed with LPS and triggered with either ( A ) ATP, ( B ) MSU or ( C ) nigericin. The diagonal seen for ATP- and MSU-triggered cells corresponds to the sum of all specks remaining at the end of the experiment.

Article Snippet: Cells were primed with 500 ng/mL ultrapure LPS (Invivogen, San Diego, CA) for 4 hours, and triggering of inflammasome complex formation was performed by addition of either 5 mM ATP (Merck, Darmstadt, Germany), 100 μg/mL MSU (Invivogen, San Diego, CA) or 10 μM nigericin (Invivogen, San Diego, CA).

Techniques:

Journal: Scientific Reports

Article Title: Time-lapse image analysis reveals trigger-dependent differences in ASC speck lifetime in the NLRP3 inflammasome

doi: 10.1038/s41598-026-50936-x

Figure Lengend Snippet: Total number of specks present and number of specks formed at each timepoint. ASC-GFP-specks in PMA-differentiated, LPS-primed THP-1-ASC-GFP cells were automatically detected and tracked. The total number of ASC-GFP-specks present at each timepoint ( A ) and number of ASC-GFP-specks formed at each timepoint ( B ) after triggering speck formation with either ATP, MSU or nigericin. Data is the sum of all fields of view from all experimental replicates ( n = 5).

Article Snippet: Cells were primed with 500 ng/mL ultrapure LPS (Invivogen, San Diego, CA) for 4 hours, and triggering of inflammasome complex formation was performed by addition of either 5 mM ATP (Merck, Darmstadt, Germany), 100 μg/mL MSU (Invivogen, San Diego, CA) or 10 μM nigericin (Invivogen, San Diego, CA).

Techniques: