naive cd4 t cell isolation kit  (Miltenyi Biotec)

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: iScience

Article Title: Fluorescence tracking Treg movement identifies anti-CCR8 and radiation as a therapeutic combination

doi: 10.1016/j.isci.2025.114572

Figure Lengend Snippet: Tregs traffic from the tumor to the TdLN at higher proportions and are equally susceptible to radiation as non-Treg CD4 T cells (A) Kaede mice were injected with either MC38, MOC1, or MOC2 cancer cells subcutaneously. Tumors were photoconverted on day 14 via ultraviolet light. TdLNs were harvested on day 3 post-photoconversion. All cell populations were pre-gated on live singlets. (B) Photoconverted CD8 T cells were defined as CD3 + CD4 − CD8 + Red + . Photoconverted Tregs were defined as CD3 + CD8 − CD4 + CD25 + Red + . Photoconverted non-Treg CD4 T cells were defined as CD3 + CD8 − CD4 + CD25 − Red + . (C) Frequencies of photoconverted CD8 T cells, Tregs, and non-Treg CD4 T cells were quantified via flow cytometry. (D) Mice were injected with MC38 cancer cells subcutaneously. Tumors were treated with 12 Gy RT on day 14 and harvested for analysis via flow cytometry on day 1–3 post-RT. Non-Treg CD4 T cells and Tregs frequencies as a percentage of live cells were quantified. (E) MC38-bearing Kaede mouse tumors were photoconverted on day 14 followed by immediate treatment with 12 Gy RT. (F) TdLNs were harvested on day 1–3 and percentages of photoconverted non-Treg CD4 T cells and Tregs were analyzed via flow cytometry. A total of 5–6 mice were analyzed per group. Data are represented as mean ± SD. Statistics were performed using an unpaired Student’s t test between groups. ns = not significant, ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05.

Article Snippet: CD4/CD8 (TIL) MicroBeads, mouse , Miltenyi Biotec , Cat #: 130-116-480.

Techniques: Injection, Flow Cytometry

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