Journal: EMBO Molecular Medicine

Article Title: Mitochondrial damage drives T-cell immunometabolic paralysis after major surgery

doi: 10.1038/s44321-025-00324-1

Figure Lengend Snippet: ( A ) Flow cytometric analysis (FCA) of CD25 (left) and CD69 (right) staining and quantification in CTL, n = 6/11 individual patients. ( B ) FCA of lymphocyte-activation gene 3 (LAG3), programmed cell death protein 1 (PD1), and T-cell immunoglobulin and mucin-domain containing-3 (TIM3) staining and quantification as indicated, n = 6/4 (not activated/+CD3/CD28) individual patients. P values (left to right): 0.2842, 0.2188, 0.6875, 0.5494, 0.1250, and >0.9999. ( C ) Quantification of relative cytotoxicity using a flow cytometric killing assay, n = 5 individual patients. ( D ) Quantification of caspase-3 activity reporter using a CTL-K562 killing assay, n = 4 individual patients. Data were represented as mean ± SEM with dots indicating individual values ( A , B ) or as individual data points ( C , D ). P values: as indicated, paired t -test or Wilcoxon matched-pairs signed-rank test, as appropriate.

Article Snippet: CD69 FITC , Miltenyi , 130-112-801.

Techniques: Staining, Activation Assay, Activity Assay

Journal: EMBO Molecular Medicine

Article Title: Mitochondrial damage drives T-cell immunometabolic paralysis after major surgery

doi: 10.1038/s44321-025-00324-1

Figure Lengend Snippet: ( A ) FCA of MitoTracker green and quantification in isolated mitochondria from CTL, n = 7 individual patients. ( B ) FCA of JC1 green and quantification of red/green fluorescence in isolated mitochondria from CTL, n = 4 individual patients. ( C ) Immunoblot of OXPHOS complexes using isolated mitochondria of CTL and normalized densitometric quantification of complex V, n = 5 individual patients. I–V complex I–V; H heat shock protein 60 (HSP60). Subunits detected by the antibody cocktail: complex I: subunit NDUFB8; complex II: subunit 30 kDa (SDHB); complex III: subunit Core 2 (UQCRC2); complex IV: subunit II (COXII); complex V: ATP synthase subunit alpha (ATP5). ( D , E ) Representative immunoblot of mitochondrial fusion and fission proteins of isolated mitochondria of CTL ( D ) and normalized densitometric quantification ( E ) of the respective proteins, n = 5/4/3 individual patients. ( F ) Representative projections of 3D image stacks of CTL stained with MitoTracker (MT, green, left) and the respective skeleton images (white, right), generated using mitochondrial analyzer, time points as indicated, representative of six individual patients. ( G ) Quantification of mitochondrial count per cell, n = 29 cells from three individual patients. ( H ) Quantification of mitochondrial length (left) and mean mitochondrial area using mitochondrial analyzer (right), n = 180/170 mitochondria from 14/22 cells from three individual patients (length) and n = 50 cells from three individual patients (area). ( I ) Representative projection of 3D image stacks (two per time point) of translocated mitochondria to the IS in CTL as indicated by MitoTracker (MT) Deep Red and CD3 FITC staining, and quantification of mitochondrial translocation to the CTL IS, time points as indicated, n = 14 from 5 individual patients. If not stated otherwise, data were represented as mean ± SEM. P values as indicated, paired t -test or Wilcoxon matched-pairs signed-rank test ( A – E ), and unpaired t -test or Mann–Whitney test ( G – I ), as appropriate. .

Article Snippet: CD69 FITC , Miltenyi , 130-112-801.

Techniques: Isolation, Fluorescence, Western Blot, Staining, Generated, Translocation Assay, MANN-WHITNEY

Journal: EMBO Molecular Medicine

Article Title: Mitochondrial damage drives T-cell immunometabolic paralysis after major surgery

doi: 10.1038/s44321-025-00324-1

Figure Lengend Snippet: ( A ) FCA of MitoTracker green and quantification in CD8 + CTL, n = 7 individual patients. ( B ) Representative Seahorse OCR plot. ( C ) Fold change of basal OCR, maximum OCR, spare respiratory capacity (SRC) and ATP production, n = 6 individual patients. ( D ) Quantification of mitochondrial length, n = 92 mitochondria from 28 cells from three individual patients. ( E ) Quantification of mitochondrial count per cell, n = 30/26 cells from three individual patients. ( F ) Representative confocal microscopy image of mitochondria translocated to the TCR in CTL as indicated by MitoTracker (MT) Deep Red and CD3 FITC staining, T2 image as depicted in Fig. , upper right image. ( G ) Quantification of mitochondrial translocation in proximity to the CTL IS, n = 12 from four individual patients. ( H ) Representative impedance plot and ( I ) quantification of relative CTL cytotoxicity using ECIS, n = 6 individual patients. If not stated otherwise, data are represented as mean ± SEM. P values as indicated, one-sample t -test ( C ), paired t -test or Wilcoxon matched-pairs signed-rank test ( A – C, H , I ), and unpaired t -test or Mann–Whitney test ( D – G ), as appropriate. .

Article Snippet: CD69 FITC , Miltenyi , 130-112-801.

Techniques: Confocal Microscopy, Staining, Translocation Assay, MANN-WHITNEY