CD8b Monoclonal Antibody (eBioH35-17.2 (H35-17.2)), eBioscience™
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Staining of BALB/c splenocytes with 0.25 µg of Rat IgG2b Isotype Control Purified (Product # 14-4031-82) (open histogram) or 0.25 µg of Purified Anti-Mouse CD8b Purified (filled histogram) followed by Anti-Rat IgG FITC (Product # 11-4811-85). Cells in the lymphocyte gate were used for analysis.
Published figure using CD8b monoclonal antibody (Product # 14-0083-82) in Flow Cytometry
Figure 1 NDV induces upregulation of ICOS in the tumour microenvironment. Bilateral flank B16-F10 melanoma-bearing mice were treated intratumorally with NDV. ( a ) Treatment scheme. ( b , c ) Representative flow cytometry plots of tumour-infiltrating CD4 + and CD8 + cells isolated from the virus-injected (right) and distant (left) tumours, gated on the total CD45 + cell population. ( d , e ) Absolute number of tumour-infiltrating CD8 + , CD4 + FoxP3 - (Tcon) and CD4 + FoxP3 + (Treg) lymphocytes isolated from the virus-injected ( d ) and distant ( e ) tumours. Relative percentages of Tregs are shown in the rightmost panels. ( f ) Absolute numbers of lymphocytes (left) and dendritic cell subsets (right) in the lymph nodes draining virus-injected tumours. ( g ) Expression of immune-related genes in NDV-injected tumours. Orange indicates high expression and blue indicates low expression. ( h ) Volcano plot of gene expression in NDV-injected tumours. Genes related to T-cell co-stimulatory function are indicated. ( i , j ) Upregulation of ICOS on the surface of tumour-infiltrating CD8 and CD4 + FoxP3 - cells in the NDV-injected ( i ) and distant ( j ) tumours. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001, NS, non-significant. mDC, myeloid dendritic cell; pDC, plasmacytoid dendritic cell; Tcon, conventional CD4 T cell (CD4 + FoxP3 - ); Treg, regulatory T cell (CD4 + FoxP3 + ). ( b - j ) Representative data of three experiments with 5-10 animals per group. Data with error bars
Figure 4 The miR-200/ZEB1 axis controls tumor metastasis through regulating CD8 + TILs ( a , b ) FACS analysis of ( a ) CD8 + TIL frequency; ( b ) PD1 and TIM3 marker expression on CD8 + T cells from 393P_vector and 393P_ZEB1 (n = 5), as well as 344SQ_vector and 344SQ_miR-200 (n = 10) primary tumors. Analysis was done 2 weeks post-cancer cell injection. ( c , d ) ( c ) Intratumoral Ki67 + CD8 + T cells; ( d ) granzyme B (GzB) + CD8 + T cells in 344SQ_vector or 344SQ_miR-200 primary tumors 6 weeks post-subcutaneous injection of cancer cells into 129/Sv mice. Representative Ki67 or GzB staining in an individual tumor sample is shown on the left, and mean Ki67 + or GzB + populations of gated CD8 + T cells in total T cells are shown on the right (n = 5). ( e ) CD8 + T cell depletion results in tumor growth and metastasis in mice (n = 5) that received subcutaneous tumor cell injections. No treatment (344SQ_vector (Vector)), IgG (344SQ_miR-200 + IgG control), or Ab (344SQ_miR-200 + anti-CD8 Ab). The analysis was done 6 weeks post-injection. ( f ) Relative abundance of CD8 + T cells in the tumor (left) or lung (right) from 129/Sv mice (n =5) with syngeneic control 344SQ tumors (Vector), 344SQ_miR-200 tumors with control IgG treatment (IgG) or anti-CD8 antibody treatment (Ab). ( g ) Lung metastases of 344SQ_vector (Vector) and 344SQ_miR-200 (miR-200) tumors in wild-type (WT) or 129/Sv Rag2 -/- ( Rag2 -/- ) mice (n = 5). The analysis was done 6 weeks post-tumor cell subcutaneous injec
Figure 7 PD-L1 expression on tumor cells is critical to the repression of anti-tumor immunity ( a ) The knockdown (KD) efficiency of PD-L1 in LLC-JSP murine lung cancer cells measured by FACS. Representative histograms (left), and statistical analysis (right). The measurement was independently repeated at least three times. ( b ) Representative FACS histogram of PD-L1 expression on myeloid cells (CD11b + ) in PD-L1 KO or WT mice (n = 3). ( c ) Tumor growth in PD-L1 KO or WT mice (n = 6) of subcutaneously injected LLC-JSP cells (10, 000 cells with 100 mul of PBS per mouse) with differing PD-L1 knockdown (Vector, PD-L1 KD vector control; PD-L1 int , PD-L1 intermediate KD; PD-L1 hi , PD-L1 high level KD). The data is shown from two independent experiments. Data are shown as mean +- s.e.m. ( d ) FACS analysis of CD8 + TIL frequency and T cell exhaustion marker expression levels on CD8 + T cells in subcutaneous primary tumors of LLC-JSP vector control (Vector ctrl) and LLC-JSP intermediate PD-L1 knockdown (Intermediate KD) in PD-L1 WT (WT) and PD-L1 KO (KO) mice (n = 5, from two independent experiments). Analysis was done 3 weeks post-tumor cell injection (20, 000 cells with 100 mul of PBS per mouse). t -test was used to analyze the data. p < 0.0001. ( e ) PD-L1 expression levels on LLC-JSP-shPD-L1 cells (the high knockdown efficiency cells) after reconstitution of PD-L1. The measurement was independently repeated at least three times. Control, LLC-JSP-shPD-L1 + vector control; PD
Figure 3 CD8 + TILs determine the metastatic potential in lung adenocarcinoma models ( a ) CD8 + T cells measured by flow cytometric analysis in single-cell suspensions prepared from tumor-bearing lungs of 8- to 12- month-old K-ras LA1/+ ( K-ras ) and K-ras LA1/+ p53 R172HDeltaG/+ ( KP ) spontaneous mouse model (n = 5). The analysis was independently repeated at least three times. t -test, p = 0.0006. ( b ) The results of a FACS analysis of CD8 + TILs in 344SQ and 393P primary tumors isolated 2 weeks after subcutaneous tumor cell injection into 129/Sv mice (n = 10). The results contain the data from two independent experiments. t -test, p < 0.0001. ( c ) Representative flow cytometric plot of CD8 + TIL numbers from 393P tumor-bearing 129/Sv mice (n = 5) treated with anti-CD8 or IgG control antibodies (200 mug, intraperitoneally; twice weekly for 2 weeks beginning on day 1 after a subcutaneous cancer cell injection). ( d ) Lung metastases in WT or 129/Sv Rag2 -/- mice (n = 5) injected subcutaneously with 344SQ or 393P cells and necropsied 5 weeks later. The analysis was independently repeated twice. t -test, p < 0.0001. ( e ) CD8 + T cells isolated from 129/Sv mice were adoptively transferred to syngeneic 129/Sv Rag2 -/- mice (n = 5). Representative FACS histograms depict the levels of CD8 + T cells in 344SQ tumors and lungs of the reconstituted mice with reconstituted CD8 + T cells versus those in the controls.
Figure 5 Genetic targeting of PD-L1 expression on cancer cells reverses the CD8 + TIL dysfunction and suppresses metastasis ( a ) Cell surface expression of PD-L1 on 344SQ PD-L1 knockdown (344SQ-shPD-L1) vs 344SQ scramble control (344SQ-scr) cells by FACS (red line, isotype control staining; blue line, anti-PD-L1 staining). The measurement was independently repeated at least three times. ( b ) Primary tumor mass (top left) and lung metastases (top right) in 129/Sv mice (n = 10) injected subcutaneously with 344SQ-shPD-L1 or 344SQ-scr cancer cells. Micrometastases (bottom) observed in hematoxylin and eosin-stained lung tissue sections are indicated by yellow arrows. Scale bar, 2mm. Samples were obtained 6 weeks post-injection. The data from two independent experiments were pooled. Data are shown as mean +- s.e.m. t -test was used to analyze. P values are shown in the graphs. ( c , d ) FACS analysis of ( c ) surface PD1, LAG3, and TIM3 marker expression levels on CD8 + T cells; ( d ) CD8 + TIL frequency for primary tumors in 129/Sv mice (n = 5) injected subcutaneously with 344SQ-shPD-L1 (shPD-L1) or 344SQ-scr control (Control) cancer cells and necropsied 2 weeks later. The analyses were independently repeated three times. Data are shown as mean +- s.e.m. t -test was used to analyze. P values are shown in the graphs.
Figure 9 Double positive thymocytes are reduced in favor of an increased CD4 + single positive population in the Snai2 and Snai3 DKO. FACS analysis was performed to assess T cell populations in the thymus (A), peripheral blood (B), and spleen (C). Cells were assayed for CD4 and CD8 cell surface staining. DP = CD4 + CD8 + double positive cells, CD4 = CD4 + single positive, CD8 = CD8 + single positive, Results are presented as a percentage of total cells analyzed. One-way ANOVA with Bonferroni post hoc test: * p < 0.05, *** p < 0.001.
Published figure using CD8b monoclonal antibody (Product # 14-0083-82) in Flow Cytometry
Description: The eBioH35-17.2 monoclonal antibody reacts with the mouse CD8 beta molecule. The CD8 beta chain associates with the CD8 alpha chain to form the CD8 alpha/beta heterodimer expressed on the surface of a majority of thymocytes, and on peripheral cytotoxic alpha beta TCR T cells. CD8 binds to MHC class I and plays a role in T cell development and activation of mature T cells.
Applications Reported: This eBioH35-17.2 (H35-17.2) antibody has been reported for use in flow cytometric analysis, immunoprecipitation, and immunohistochemical staining of frozen tissue sections. Applications Tested: This eBioH35-17.2 (H35-17.2) antibody has been tested by flow cytometric analysis of mouse splenocytes and thymocytes. This can be used at less than or equal to 0.5 µg per test. A test is defined as the amount (µg) of antibody that will stain a cell sample in a final volume of 100 µL. Cell number should be determined empirically but can range from 10^5 to 10^8 cells/test. It is recommended that the antibody be carefully titrated for optimal performance in the assay of interest. Purity: Greater than 90%, as determined by SDS-PAGE. Aggregation: Less than 10%, as determined by HPLC. Filtration: 0.2 µm post-manufacturing filtered.
The CD8B antigen is a cell surface glycoprotein found on most cytotoxic T lymphocytes that mediates efficient cell-cell interactions within the immune system. The CD8 antigen, acting as a coreceptor, and the T-cell receptor on the T lymphocyte recognize antigens displayed by an antigen presenting cell (APC) in the context of class I MHC molecules. The functional coreceptor is either a homodimer composed of two alpha chains, or a heterodimer composed of one alpha and one beta chain. Both alpha and beta chains share significant homology to immunoglobulin variable light chains. This gene encodes the CD8 beta chain isoforms. Multiple alternatively spliced transcript variants encoding distinct membrane associated or secreted isoforms have been described. A pseudogene, also located on chromosome 2, has been identified.