Anti-CD8 alpha/Cd8a Antibody Picoband®

Western blot analysis of CD8 alpha using anti-CD8 alpha antibody (A02236-1).
Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 50ug of sample under reducing conditions.
Lane 1: rat spleen tissue lysate,
Lane 2: rat thymus tissue lysate.
After Electrophoresis, proteins were transferred to a Nitrocellulose membrane at 150mA 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-CD8 alpha antigen affinity purified polyclonal antibody (Catalog # A02236-1) 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 at a dilution of 1:10000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for CD8 alpha at approximately 36KD. The expected band size for CD8 alpha is at 26KD.

Immunohistochemical analysis of the CD8a-positive area in the injured spinal region 7 days after SCI. Bar: 40 µm.
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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.
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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.
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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.
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Targeting USP7 inhibits murine M2 phenotype and function in vitro . (A) The mRNA expression of common genes related to DUBs was detected by RT-PCR in M0, M1 (LPS/IFN-γ M1), and M2 (IL-4/13 M2 and IL-10 M2) induced from ANA-1. ( B ) The mRNA expression of USP7 in MΦs, M1, and M2 induced from BMDMs was detected by RT-PCR. ( C ) Western blotting showing the expression of USP7 in MΦ, M1, and M2 induced from BMDMs. ( D-E ) Flow cytometry analyses of the expression of CD206 in IL-4/13-induced BMDM M2 cells, which had been treated with P5091 (5 μM or 10 μM) for 24 h. Data are presented as the mean ± SEM (n = 3). ( F ) Detection of the expression of USP7 in BMDMs, which were transfected with either NC- or USP7-siRNA by western blotting. ( G ) The expression of CD206 in BMDMs of NC-siRNA and USP7-siRNA group was detected by flow cytometry. ( H-I ) Flow cytometry analyses of CFSE expression on the surface of CD8 + T cells in the presence of conditioned medium from either DMSO- or P5091- (5 μM or 10 μM) treated IL-4/13-induced BMDM M2 cells. Data are presented as the mean ± SEM (n = 3).
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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.
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Targeting USP7 inhibits tumor growth and induces local anti-tumor immunity in vivo . ( A ) Harvested and photographed tumors in the P5091 and the Control group. ( B ) Lewis tumor growth following P5091 or Vehicle treatment in vivo. Data are presented as the mean ± SEM (n = 6 per group). ( C ) Spider diagram of the tumor volume growth in each mouse from the P5091 group and the Control group. ( D ) Gating strategy for detection of the TAMs by flow cytometry. ( E-G ) Proportions of M1 ( E ) and M2 ( F ), and M1/M2 ratio ( G ) in the TME of the P5091 group and the Control group. ( H-M ) Percentages of MDSC ( H ), Treg ( I ), CD4 + T ( J ), CD8 + T ( K ), Th1 ( L ), and CTLs ( M ) within the TME in each group. Data are presented as the mean ± SEM (n = 7) for ( E-M ).
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Targeting USP7 activates local and systemic anti-tumor immunity. ( A ) Multicolor immunofluorescence detection of TAMs and CTLs in the TME of the P5091 group and the Control group. ( B-F ) Cytokines IFN-γ ( B ), TNF-α ( C ), IL-2 ( D ), IL-5 ( E ), and IL-6 ( F ) in the TME of each group were detected by Mul-Analyte Flow Assay Kit. ( G-K ) Proportion of Treg ( G ), CD4 + T ( H ), CD8 + T ( I ), Th1 ( J ), and CTLs ( K ) in the spleen of each group. Data are presented as the mean ± SEM (n = 7) for ( B-K ).
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Targeting USP7 can activate the p38 MAPK pathway to reprogram TAMs. ( A ) The strategy for sorting TAMs in TME by flow cytometry. ( B ) Heat maps illustrating the differentially expressed M1- and M2-related genes in TAMs between the P5091 group and the Control group based on the results of RNA sequencing. ( C ) RT-PCR further verifying the differentially expressed genes of sorted TAMs in each group. Data are presented as the mean ± SEM (n = 3). ( D ) KEGG analysis identifying 20 most obviously enriched pathways based on the differentially expressed genes of the two groups. ( E ) Western blotting detection of the expression of JNK, p-JNK, ERK1/2, p-ERK1/2, p38, p-p38, and β-actin in IL-4/13-BMDM M2 cells treated with P5091 (10 µM) at the indicated time points. ( F ) Flow cytometry analysis of CFSE expression on the surface of CD8 + T cells in the presence of conditioned medium of IL-4/13-induced BMDM M2 cells from various indicated treatments. Treatments indicated: DMSO stimulation, P5091 (10 µM) stimulation, P5091 (10 µM) stimulation in the presence of inhibitors of p38 (SB203580, 10 µM), JNK (SP600125, 10 µM), Erk1/2 (U0126-EtOH, 10 µM). Data are presented as the mean ± SEM (n = 4).
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ASNS shapes the immune landscapes in metastatic TdLN and primary tumor site. Figure 5A-C. LN metastasis model was conducted on C57BL/6 mice with LLC-ASNS WT(n=6), ASNSC2A overexpression cells(n=6) and control group(n=6), primary tumor and popliteal lymph nodes were isolated at the end of the experiment, and primary tumor volume(B) and TdLN volume/tumor volume(C) was measured and analyzed. 5D-F. (D) Representative FACS profiles of CD8+T cells are shown. The percentage(E) and number(F) of CD8+ subset in TIL cells isolated from primary tumor is shown. 5G-I. (G) Representative FACS profiles of the co-expression pattern of CD44 and CD62L, or the co-expression pattern of TCF-1and TOX in CD8+ T cells are shown. The number(H) and percentage(I) of CD44+, CD44+CD62L+, CD44+CD62L-, Tsl and TTSM subset in CD8+T cells isolated from TdLN is shown. 5J-L. (J) Representative FACS profiles of the co-expression pattern of CD44 and CD62L, or the co-expression pattern of TCF-1and TOX in CD8+ T cells are shown. The number(K) and percentage(L) of CD44+, CD44+CD62L+, CD44+CD62L-, Tsl and TTSM subset in CD8+T cells isolated from primary tumor is shown.
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ASNS-high-expression metastases generated lymphocyte niches enriched with activated T cells, memory T cells, Tsl and TTSM. Figure 6A-C. Representative immunofluorescence staining images of metastatic TdLNs from LN metastasis model. 6D. The number of CD8+ T cells in the metastasis locations within TdLNs (ASNSWT, n=6, ASNSC2A, n=5, and EV, n=4). 6E-F. The number(E) and percentage(F) of CD44+CD8+T cells among all CD8 T cells in the metastasis locations within TdLNs (ASNSWT, n=6, ASNSC2A, n=5, and EV, n=4). 6G-H. The number(G) and percentage(H) of Tsl cells among all CD8 T cells in the metastasis locations within TdLNs (ASNSWT, n=6, ASNSC2A, n=5, and EV, n=4). 6I-J. The number(I) and percentage(J) of TTSM cells among all CD8 T cells in the metastasis locations within TdLNs (ASNSWT, n=5, ASNSC2A, n=4, and EV, n=3). 6K. Quantitative estimates of the distance from ova+ to CD8+CD44+CD62L+TCF+(TTSM) (ASNSWT, n=6, ASNSC2A, n=5, and EV, n=4). 6L-M. Representative immunofluorescence staining images of metastatic TdLNs from NSCLC patients. 6N-O. The number(C) and percentage(D) of CD8+ T cells in the metastasis locations within TdLNs(ASNS high group, n=7, and ASNS low group, n=6). 6P-Q. The number (E) and percentage(F) of CD45RO+CD8+ T cells in CD8+T cells in the metastasis locations within TdLNs(ASNS high group, n=7, and ASNS low group, n=6). 6R-S. The number (G) and percentage(H) of Tsl cells in CD8+T cells in the metastasis locations within TdLNs(ASNS high group, n=7, and ASNS low group, n=6). 6T-U. The number (I) and percentage(J) of TTSM cells in CD8+T cells in the metastasis locations within TdLNs(ASNS high group, n=7, and ASNS low group, n=6).
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IHC analysis of CD8 alpha using anti-CD8 alpha antibody (A02236-1).
CD8 alpha was detected in paraffin-embedded section of mouse spleen tissue . Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti-CD8 alpha Antibody (A02236-1) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC)(Catalog # SA1022) with DAB as the chromogen.

IHC analysis of CD8 alpha using anti-CD8 alpha antibody (A02236-1).
CD8 alpha was detected in paraffin-embedded section of rat spleen tissue . Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti-CD8 alpha Antibody (A02236-1) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC)(Catalog # SA1022) with DAB as the chromogen.

Flow Cytometry analysis of mouse spleen tissues using anti-CD8 alpha antibody (A02236-1).
Overlay histogram showing mouse spleen tissues stained with A02236-1 (Blue line). The tissues were fixed with 4% paraformaldehyde and blocked with 10% normal goat serum. And then incubated with rabbit anti-CD8 alpha Antibody (A02236-1, 1 μg/1x10⁶ cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-rabbit IgG (BA1127, 5-10 μg/1x10⁶ cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was rabbit IgG (1 μg/1x10⁶) used under the same conditions. Unlabelled sample without incubation with primary antibody and secondary antibody (Red line) was used as a blank control.