Journal: Biofabrication

Article Title: Collagen hydrogel tube microbioreactors for cell and tissue manufacturing

doi: 10.1088/1758-5090/ae2718

Figure Lengend Snippet: Culturing Adherent HEK 293 T Cells in ColTubes. Phase-contrast and Live/Dead staining images of adherent HEK 293 cells (A) and suspension 293 T 17SF cells (B) in ColTubes.

Article Snippet: Suspension 293 T 17SF cells (#ACS-4500, ATCC) in ColTubes were cultured in HyCellTransFx-H Medium (Cytiva).

Techniques: Staining, Suspension

Journal: Biochemistry and Biophysics Reports

Article Title: NLRP3 inflammasome regulates Th17/Treg cell balance in experimental autoimmune myocarditis

doi: 10.1016/j.bbrep.2026.102447

Figure Lengend Snippet: Inhibition of NLRP3 shifted the differentiation of naïve CD4 + T cells toward a Treg phenotype in vitro. ( a ) Flow cytometry plots identifying Th17 and Treg populations in EAM versus MCC950-treated groups; ( b ) Bar graphs quantifying Th17 and Treg frequencies in EAM versus MCC950-treated groups; ( c ) Secreted IL-17 and IL-10 protein levels in culture supernatants; ( d ) RORγt and Foxp3 mRNA expression levels. Data are presented as mean ± SD, n = 3. NS indicates no statistical significance; ∗p < 0.05, ∗∗p < 0.01.

Article Snippet: Naive CD4 + T cells were isolated using the specific naive CD4 + T cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany)and cultured in 24-well plates at 1 × 10 6 cells/well in complete DMEM medium supplemented with 10 % FBS and 1 % penicillin-streptomycin.

Techniques: Inhibition, In Vitro, Flow Cytometry, Expressing

Journal: Blood Advances

Article Title: Functional recovery of NK cells after T-cell replete haploidentical HSCT: delayed licensing and poor anti-acute myeloid leukemia activity

doi: 10.1182/bloodadvances.2025017707

Figure Lengend Snippet: Evolution of licensing ligand expression and licensing phenotype after h-HSCT. (A) Normalized expression of total HLA class I, HLA-C, and HLA-E over time. Median fluorescence intensity (MFI) normalized to isotype control for total HLA class I, HLA-C, and HLA-E on total lymphocytes, T cells (CD3 + CD56 − CD19 − ), and NK cells (CD3 − CD56 + ) at each time point. Data are presented as box plots with overlaid individual values and density plots. Comparisons between time points were performed using Wilcoxon test. (B) Study design; peripheral blood samples were collected from h-HSCT recipients at the indicated time points. Donor (D) and recipient (R) combinations were identified based on the presence (+) or absence (−) of HLA-C C1 or C2 ligands and degranulation of NKG2A + KIR − , NKG2A − KIR − , NKG2A − KIR2DL1 single+ , and NKG2A − KIR2DL2/3 single+ NK cell subsets was evaluated after coculture with the HLA class I−negative 721.221 cell line. (C) Correlation between degranulation of KIR − NKG2A + NK cells and normalized MFI of HLA-E (on total lymphocytes) at each time point. Spearman correlation coefficients and P values are indicated. Linear regression with 95% CI is shown. (D-E) Paired comparison of degranulation of NKG2A − KIR − and NKG2A − KIR2DL1 single+ (D) or KIR2DL2/3 single+ (E) NK cell subsets according to D and R HLA-C combinations at each time point. Data are presented as paired comparisons with individual values. Paired comparisons were performed using Wilcoxon signed-rank test. ∗ P < .05; ∗∗ P < .01; ∗∗∗ P < .001; ∗∗∗∗ P < .0001.

Article Snippet: NK cell cytotoxic potential was evaluated by CD107a expression after 5-hour coculture with 4 hematological malignancy cell lines: H9 (T-cell non-Hodgkin lymphoma, ATCC HTB-176), MOLT-4 (T-cell acute lymphoblastic leukemia, ATCC CRL-1582), KG-1 (acute myeloblastic leukemia [AML], ATCC CCL-246), and NB4 (AML, DSMZ ACC 207).

Techniques: Expressing, Fluorescence, Control, Comparison

Journal: Inflammation Research

Article Title: mTOR pathway mediates the endoplasmic reticulum stress -apoptosis of CD4+ T cell through inhibiting autophagy flux in sepsis

doi: 10.1007/s00011-025-02114-4

Figure Lengend Snippet: CD4 + T cells apoptosis is increased and correlated with ER stress. A – D The rate of apoptosis of CD4 + T cells was measured by the ratio of Annexin V-positive and PI-positive/negative CD4 + T cells. E – F The expression level of bax, bcl2, and caspase 3 were examined by Western blotting. G – H The expression level of GRP78and CHOP, the marker of ER stress was measured by western blotting. I – K The microstructure images of ER of CD4 + T cells in WT, WT + CLP, and CLP + 4-PBA mice were observed with electron microscopy. Green arrows represent normal-sized ER. Yellow arrows represent dilation and vesiculation of the ER. Densitometric quantification for expression of protein was normalized to ACTIN protein level. Means ± standard deviations (SDs) of four mice per group are shown. It was deemed statistically significant when P < 0.05. * P < 0.05.* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: The splenocyte suspension was incubated with CD4 + antibody (CD4 + T cells isolation Kit, mouse Miltenyi, 130–104-454), washed, and passed through a separation column for negative screening, and finally centrifuged to obtain CD4 + T cells.

Techniques: Expressing, Western Blot, Marker, Electron Microscopy

Journal: Inflammation Research

Article Title: mTOR pathway mediates the endoplasmic reticulum stress -apoptosis of CD4+ T cell through inhibiting autophagy flux in sepsis

doi: 10.1007/s00011-025-02114-4

Figure Lengend Snippet: The role of mTOR in ER stress-induced CD4 + T cells apoptosis. A – C Proteins of mTOR pathway, including mTOR, P -mTOR, downstream effectors p70s6k, p-p70s6k were examined by Western blotting. D – E The expression level of GRP78and CHOP, the marker of ER stress was measured by western blotting. F – H The expression level of bax, bcl2, and caspase 3 were examined by Western blotting. Means ± standard deviations (SDs) of four mice per group are shown. It was deemed statistically significant when P < 0.05. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: The splenocyte suspension was incubated with CD4 + antibody (CD4 + T cells isolation Kit, mouse Miltenyi, 130–104-454), washed, and passed through a separation column for negative screening, and finally centrifuged to obtain CD4 + T cells.

Techniques: Western Blot, Expressing, Marker

Journal: Inflammation Research

Article Title: mTOR pathway mediates the endoplasmic reticulum stress -apoptosis of CD4+ T cell through inhibiting autophagy flux in sepsis

doi: 10.1007/s00011-025-02114-4

Figure Lengend Snippet: Deficient autophagy is observed under ER stress in sepsis and the role of mTOR on it. (A-E) With flow cytometry, the rates apoptosis of CD4 + T cells were detected in WT + CLP, mTOR KO + CLP, TSC1 KO + CLP, TSC1 KO + CLP + 4-PBA. (F, H). The expression level of LC3I/LC3II and P62, the markers of autophagy process were measured by western blotting. G , I – K Ultrastructural features of CD4 + T cells were investigated using transmission electron microscopy (TEM). In WT group, CD4 + T cells had normal morphologies, revealing baseline autophagy status. WT + CLP mice displayed increased autophagic vacuolization but no significant increase in autolysosome frequency. Large autolysosomes containing abundant contents were seen. More autophagic vacuolization and more autolysosomes were showed in mTOR KO + CLP. Autophagosomes and autolysosomes were fewer in TSC1 KO + CLP mice. Autophagosomes were double-membrane vacuoles containing cytosol or organelles (red arrow). Autolysosomes were single-membrane structures containing digested cytoplasmic components (blue arrow). Means ± standard deviations (SDs) of four mice per group are shown. It was deemed statistically significant when P < 0.05. ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: The splenocyte suspension was incubated with CD4 + antibody (CD4 + T cells isolation Kit, mouse Miltenyi, 130–104-454), washed, and passed through a separation column for negative screening, and finally centrifuged to obtain CD4 + T cells.

Techniques: Flow Cytometry, Expressing, Western Blot, Transmission Assay, Electron Microscopy, Membrane

Journal: Inflammation Research

Article Title: mTOR pathway mediates the endoplasmic reticulum stress -apoptosis of CD4+ T cell through inhibiting autophagy flux in sepsis

doi: 10.1007/s00011-025-02114-4

Figure Lengend Snippet: mTOR deletion activates autophagy to alleviate ER stress-induced apoptosis. (A-F) The proportion of apoptosis of CD4 + T cells were detected in WT + CLP, mTOR KO + CLP, CLP + Rap, mTOR KO + CLP + Baf, CLP + Baf by flow cytometry analysis. Densitometric quantification for expression of protein was normalized to ACTIN protein level. G – I The expression level of GRP78, CHOP, bax, and bcl2, the marker of ER stress and apoptosis were measured by western blotting. Data was presented as means ± standard deviations (SDs) of four mice per group are shown. It was deemed statistically significant when P < 0.05. ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: The splenocyte suspension was incubated with CD4 + antibody (CD4 + T cells isolation Kit, mouse Miltenyi, 130–104-454), washed, and passed through a separation column for negative screening, and finally centrifuged to obtain CD4 + T cells.

Techniques: Flow Cytometry, Expressing, Marker, Western Blot

Journal: Journal of Clinical Apheresis

Article Title: Suppressive CD8 + T‐Cells Are Key Cellular Mediators of Extracorporeal Photopheresis

doi: 10.1002/jca.70094

Figure Lengend Snippet: The protective effect of ECP is dependent on CD8+ T cells. For all experiments 6–8 week old female mice of the indicated strain were used. EAE was induced with the indicated peptide on Day 0 and mice were monitored daily for signs of disease. (A) ECP was performed using spleen + LN cells obtained from PLP 139‐151 immunized mice on Day 12 post immunization. 10 million cells were transferred to recipient mice on Day 0. Control mice received no cells. N = 17 mice/group. (B) ECP was performed using spleen + LN cells obtained from PLP 178‐191 immunized mice on Day 12 post immunization. 10 million cells were transferred to recipient CD8 −/− mice on Day 0. Control mice received no cells. N = 11 mice/group. (C, D) ECP was performed using spleen + LN cells obtained from PLP 178‐191 immunized mice on Day 12 post immunization. 10 million cells were transferred to recipient mice on Day 0. Control mice received no cells. On Day 14 of EAE, mice were euthanized, spleens and LNs were harvested, and cells were incubated with IL‐2 and PLP 178‐191 for 72 h. CD8+ T‐cells were isolated, and five million cells were transferred to recipients at the time of EAE induction with PLP 178‐191 . Control mice received no cells (control), the remainder of the mice received CD8+ T cells obtained from ECP treated (cells from ECP‐treated) or untreated (cells from control) mice. N = 6 mice/group. Statistics were performed using Student's t ‐test with *** indicating p < 0.001.

Article Snippet: First, bulk CD8+ T cells were isolated by positive selection using the CD8+ T Cell Isolation Kit (Miltenyi Biotec, 130‐096‐495).

Techniques: Control, Incubation, Isolation

Journal: Journal of Clinical Apheresis

Article Title: Suppressive CD8 + T‐Cells Are Key Cellular Mediators of Extracorporeal Photopheresis

doi: 10.1002/jca.70094

Figure Lengend Snippet: Suppressive CD8+ T cells are increased in patients undergoing ECP. For all experiments PBMCs were isolated from whole blood obtained from healthy donors or patients undergoing ECP. (A) Flow cytometry was performed, and CD8+ suppressor cells were identified as indicated. (B–E) CD4+ CD25− cells were isolated from PBMCs, stained with CFSE, and incubated with CD8+ cells at the indicated ratios for 5–7 days. Flow cytometry was performed to identify CFSE dilute CD4+ cells (divided) and the % suppression was calculated by comparing the divided populations to CD4+ cells that were incubated in the absence of CD8+ cells (more details in the materials and methods). (B) CD8‐suppression assays in healthy donors versus ECP patients. (C) CD8‐suppression assays in paired samples obtained prior to the start of ECP (Baseline) or after 1 month of therapy. (D, E) Allo‐suppression assays. CD4+ CD25− cells were isolated from healthy donors or ECP patients. Each donor was paired with an ECP patient to allow for direct comparison between autologous and allogenic suppression results. (D) CD4+ CD25− cells from the healthy donors were incubated with CD8+ cells from the same healthy donor (auto) or a paired ECP patient (allo). (E) CD4+ CD25− cells from the ECP patients were incubated with CD8+ cells from a paired healthy donor (allo) or the same ECP patient (auto). Suppression of cell proliferation was evaluated as described above. Statistics were performed using Student's t ‐test with * indicating p < 0.05, ** indicating p < 0.01, and *** indicating p < 0.001.

Article Snippet: First, bulk CD8+ T cells were isolated by positive selection using the CD8+ T Cell Isolation Kit (Miltenyi Biotec, 130‐096‐495).

Techniques: Isolation, Flow Cytometry, Staining, Incubation, Comparison