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: 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: bioRxiv

Article Title: Tissues Guide Dependence of Treg on the Transferrin Receptor

doi: 10.64898/2026.02.01.703140

Figure Lengend Snippet: A) To test Treg suppressive function in vivo , WT and KO Tregs isolated from CD4-Cre Tfrc floxed mice were directly compared in a colitis model. B) Body weight of recipient mice was monitored during disease development until the humane end point when the first mouse had lost more than 20% of its original body weight. Average weight loss of all mice in each group is shown. C) Colons were processed for H&E staining and pathology scores were determined by a blinded veterinary pathologist for the proximal (D), middle (E), and distal (F) regions of the colon. Data are from 2 independent experiments. One-way ANOVA with Sidak’s multiple comparisons test. G) Mesenteric lymph nodes (mLN) were processed for flow cytometry to analyze the frequencies of transferred Tregs, their Foxp3 expression (H), and Helios expression (I). J) One representative tail from both recipient mice of the WT and KO Tregs. K) Ears of recipient mice were processed for flow cytometry to count Tregs (K), Foxp3 expression (L), and quantify CD4 T cells in the skin (M). (K-M) Unpaired two-tailed Student’s t-test.

Article Snippet: WT and KO Tregs were isolated from CD4-cre + and – spleens according to the (Stem Cell Technologies) and mixed in a 1 to 3 ratio with naïve CD4 T cells isolated from WT CD45.1 mice according to the Naïve CD4 T Cell Isolation Kit (Miltenyi Biotec).

Techniques: In Vivo, Isolation, Staining, Flow Cytometry, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: Tissues Guide Dependence of Treg on the Transferrin Receptor

doi: 10.64898/2026.02.01.703140

Figure Lengend Snippet: A) Representative images of Foxp3 YFP -Cre, Tfrc floxed mice with scaly skin on the tail and paws of KO mice. B) Representative images of H&E-stained ears from littermates. Scale bar= 0.10 mm. C) Ears were processed into single cell suspensions and CD4+ and cytotoxic CD8+ T cells quantified by flow cytometry. D) IFNy+ CD4 T cells quantified by flow cytometry in the skin samples. E-G) Increased infiltration of macrophages (CD11b+F4/80+), IL-17a⁺ CD4 T cells, and neutrophils (CD11b+Ly6G+) in KO ears. H) H&E-stained lung sections. Scale bar = 0.5 mm, inset scale bar= 0.1 mm. (I) Lungs were processed into single cell suspension to quantify CD4 and CD8 T cells by flow cytometry. J) Frequency of T-bet+ Th1 cells in lungs, IFN-y producing T cells and TNF-a+ T cells. K) Th2-associated populations, Gata-3+ Th2 cells, IL-13 and IL-5 cells. Frequency of Th17 cells (L) and Foxp3+ Tregs (M) in the lungs. All statistical tests are unpaired two-tailed Student’s t-test.

Article Snippet: WT and KO Tregs were isolated from CD4-cre + and – spleens according to the (Stem Cell Technologies) and mixed in a 1 to 3 ratio with naïve CD4 T cells isolated from WT CD45.1 mice according to the Naïve CD4 T Cell Isolation Kit (Miltenyi Biotec).

Techniques: Staining, Single Cell, Flow Cytometry, Suspension, Two Tailed Test

Journal: bioRxiv

Article Title: Tissues Guide Dependence of Treg on the Transferrin Receptor

doi: 10.64898/2026.02.01.703140

Figure Lengend Snippet: WT mice were treated with ethanol (EtOH) or MC903 for 10 days to induce atopic dermatitis (AD) on the ears. A) Frequency of Foxp3+ cells of CD45+CD4+ cells in the ears of WT mice. B) Flow cytometric quantification of geometric MFI (gMFI) of CD71 expression on Tregs from the ears or pLN of WT mice. C) BioTracker Labile Iron dye quantification of free iron in live GFP+ Tregs from the ears of WT mice. D) Foxp3-EGFP-ER T2 -Cre mice were treated with tamoxifen or corn oil injections to induce deletion of Tfrc and subsequently treated with ethanol (EtOH) or MC903 for 10 days. E) On day 11, MC903 treated mice were blindly scored for scratching behavior for 10 minutes each. F) Representative images of ears from each treatment group (left). Disease severity scores were assessed by a blinded dermatologist based on Physician global assessment (PGA) score. G) Representative HE images of ears. Scale bar = 500 μm. H) Total CD45+ cells in the ears were counted by flow cytometry. I) Eosinophil counts defined as total live single cell CD45+ CD11b+ Ly6G- Siglec F+ cells. J) Monocyte counts defined as total live single cell CD45+ CD11b+ Ly6G- Siglec F- F480- CD11c-. K) CD4 T cell frequencies in MC903 treated mouse ears. L) Treg frequencies in the ears and (M) pLN. N) Free iron was quantified by flow cytometry in live GFP+ Tregs from the pLN. O) Frataxin expression of Foxp3+ Tregs was determined by flow cytometry in the pLN. A, C, E, G, H, M= Welch’s t test. B, H, I, J, L, M N, O= 2 way ANOVA.

Article Snippet: WT and KO Tregs were isolated from CD4-cre + and – spleens according to the (Stem Cell Technologies) and mixed in a 1 to 3 ratio with naïve CD4 T cells isolated from WT CD45.1 mice according to the Naïve CD4 T Cell Isolation Kit (Miltenyi Biotec).

Techniques: Expressing, Flow Cytometry, Single Cell