Anti-CD4 Antibody Picoband®

Western blot analysis of CD4 using anti-CD4 antibody (A00344-2).
Electrophoresis was performed on a 10% SDS-PAGE gel at 80V (Stacking gel) / 120V (Resolving gel) for 2 hours. The sample well of each lane was loaded with 30 ug of sample under reducing conditions.
Lane 1: mouse thymus tissue lysates,
Lane 2: mouse spleen tissue lysates.
After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-CD4 antigen affinity purified polyclonal antibody (A00344-2) at 0.5 μg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody (Catalog # BA1054) at a dilution of 1:5000 for 1.5 hour at RT. The signal is developed using an ECL Plus Western Blotting Substrate (Catalog # AR1196-200) with Tanon 5200 system. A specific band was detected for CD4 at approximately 51 kDa. The expected band size for CD4 is at 51 kDa.

IHC analysis of CD4 using anti-CD4 antibody (A00344-2).
CD4 was detected in a paraffin-embedded section of mouse spleen tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 μg/ml rabbit anti-CD4 Antibody (A00344-2) overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit (Catalog # SV0002) with DAB as the chromogen.

The effects of berberine on CD4 + and CD8 + T cell apoptosis, activation, and proliferation in vitro . (A) T cell activation assay. The percentages of (i) CD4 + CD44 + and (ii) CD8 + CD44 + T cells were determined by flow cytometry. (B) T cell proliferation assay. T cells from naïve C57BL/6 mice were labeled with CFSE and then co-stimulated with anti-CD3/CD28 Abs in the absence or presence of berberine. After 3 days of co-culture, (i) CD4 + and (ii) CD8 + T cell division was determined by flow cytometry. (C) (i) Supernatant levels of IFN-γ measured by ELISA, and (ii) mRNA expression of IFN-γ in T cells measured by qPCR. * p < 0.05, ** p < 0.01, *** p < 0.001 compared to the (Positive Control) PC group.
Index in PubMed under a CC BY license. PMID: 33732240

Berberine induces T cell apoptosis via the mitochondrial apoptosis pathway. (A) (i) KEGG functional categories of differentially expressed genes following berberine and saline treatment. The Y-axis represents the KEGG functional categories. (ii) KEGG analysis of the significantly altered signaling pathways in cell growth- and death-associated genes. The X-axis represents the rich ratio of the number of differentially expressed genes and the Y-axis represents the KEGG pathways. (iii) qPCR analysis of Bcl-2 and TNF-α mRNA expression in SPCs collected from heart transplant recipients treated with berberine or not. (B) T cell apoptosis assays in vivo . (i) SPCs and (ii) LNCs were collected at POD 7. The percentages of apoptotic CD4 + and CD8 + T cells were determined by flow cytometry (n = 3 mice/group). (C) T cell apoptosis assay in vitro . T cells from naïve C57BL/6 mice were co-stimulated with anti-CD3/CD28 Abs in the absence or presence of berberine. The percentages of apoptotic (i) CD4 + and (ii) CD8 + T cells were determined by flow cytometry. (D) Berberine activates the mitochondrial apoptosis pathway in vivo . (i) Relative protein expression of Bcl-2, Bax, cytochrome c, cleaved-caspase-3, and cleaved-PARP in SPC. (ii) β-actin was used as a loading control (n = 3 mice/group), and OD values (relative to β-actin) are presented as means ± SEMs. * p < 0.05, ** p < 0.01, *** p < 0.001 compared to the normal saline-treated group. (E) Berberine activates the mitochondrial apoptosis pathway in vitro . Relative protein expression of Bcl-2, Bax, cytochrome c, cleaved-caspase-3, and cleaved-PARP expression in CD3 + T cells. (ii) β-actin was used as a loading control; OD values (relative to β-actin) are presented as means ± SEMs. SPCs, spleen cells; LNCs, lymph node cells; POD, post-operative day. * p < 0.05, ** p < 0.01, *** p < 0.001 compared to the PC group.
Index in PubMed under a CC BY license. PMID: 33732240

Phenotypic and functional characteristics of allograft-infiltrating CD4 + or CD8 + T cells. Allografts were recovered at POD 7, and POD 100 syngeneic grafts are shown for comparison. (A) (i) Immunofluorescent staining of CD4 (red), KI67 (green), and 4′,6-diamidino-2-phenylindole (DAPI, blue) in grafts. (ii) Immunofluorescent staining of CD8 (red), KI67 (green), and DAPI in grafts (Scale bar = 200 μm; original magnification: ×200). (B) Proportion and absolute number of graft-infiltrating (i) CD4 + T cells and their expression of (ii) KI67, and proportion and absolute number of graft-infiltrating (iii) CD8 + T cells and their expression of (iv) KI67 (n = 3 mice/group). (C) Proportion of (i) IFN-γ and (ii) cleaved-caspase-3 in graft-infiltrating CD3 + T cells (n = 3 mice/group). (D) (i) Cleaved-caspase-3 and cleaved-PARP protein expression in grafts. Myocardial cell apoptosis co-immunofluorescence staining and expression of (ii) cleaved-caspase-3 and (iii) cleaved-PARP (n = 3 mice/group). (E) Relative mRNA expression of IFN-γ , IL-6, IL-10 , Foxp3 , and FasL in grafts measured by qPCR (n = 3 mice/group). SPCs, spleen cells; LNCs, lymph node cells; POD, post-operative day. * p < 0.05, ** p < 0.01, *** p < 0.001 compared to the normal saline-treated group.
Index in PubMed under a CC BY license. PMID: 33732240

Effect of berberine treatment on the T cell immune responses to cardiac allografts. (A) MLR responses. Recipient SPCs were isolated at POD 7 (responders) and mitomycin C-treated naïve BALB/c SPCs (stimulators) were co-cultured for 3 days (n = 3 mice/group). Total SPCs were isolated at PDO 7, and the absolute numbers of (B) SPCs were determined by flow cytometry. (C) The percentage of CD4 + Foxp3 + Treg cells was determined by flow cytometry. Syngeneic recipients are shown for comparison (n = 3 mice/group). (D) The spleens of recipient mice were harvested and weighed at POD 7 (n = 5 mice/group). (E) The absolute numbers of CD4 + and CD8 + T cells SPCs (n = 3 mice/group). (F) SPCs and (G) LNCs were isolated at POD 7, and the percentage of CD4 + and CD8 + T cells was determined by flow cytometry. Syngeneic recipients are shown for comparison (n = 3 mice/group). (H) CD44 + CD69 + T cell activation assays. SPCs were isolated at POD 7, and the percentages of (i) CD4 + CD44 + CD69 + and (ii) CD8 + CD44 + CD69 + T cells were determined by flow cytometry (n = 3 mice/group). (I) LNCs were isolated at POD 7, and the percentages of (i) CD4 + CD44 + CD69 + and (ii) CD8 + CD44 + CD69 + T cells were determined by flow cytometry (n = 3 mice/group). (J) Serum plasma levels of proinflammatory cytokines. Peripheral blood was collected at POD 7, and (i) IFN-γ, (ii) IL-6, and (iii) TNF-α plasma levels were measured by ELISA (n = 3 mice/group). SPCs, spleen cells; LNCs, lymph node cells; MLR, mixed lymphocyte reaction; POD, post-operative day. * p < 0.05, ** p < 0.01, *** p < 0.001 compared to the normal saline-treated group.
Index in PubMed under a CC BY license. PMID: 33732240