Cd8 clone 4sm15

Manufactured by Thermo Fisher Scientific

The CD8 (clone 4SM15) is a laboratory reagent used for the identification and enumeration of CD8+ T cells in flow cytometry applications. It binds specifically to the CD8 cell surface receptor, which is expressed on a subset of T lymphocytes.

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CD8 (4SM15) (2 citations) (clone 4SM15, (2 citations)

8 protocols using cd8 clone 4sm15

Multiplex Immunostaining for Tumor Analysis

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Immunostaining for Ki-67 (Vector), HER2 (AB16901; Abcam), and STAT1 (AB47425; Abcam) was performed as previously described^{6 (link)}. Secondary antibodies (AF488 AffiniPure donkey anti-mouse IgG, Cy3 AffiniPure donkey anti-rabbit IgG) were from Jackson ImmunoResearch Laboratories. Images were acquired with a Yokogawa spinning disk confocal on an inverted Nikon Ti fluorescence microscope using MetaMorph image acquisition software, and 3–5 fields were analyzed per tumor. Image analysis was performed using a semi-automated in-house platform (NIH ImageJ).
Immunofluorescence for FoxP3 (clone FJK-16s; eBioscience), CD8 (clone 4SM15; eBioscience), and Ki-67 (clone SP6; Thermo Scientific) was performed as previously described^{21 (link)}. Secondary antibodies (AF488 donkey anti-rabbit IgG, AF647 goat anti-rat IgG) were from Life Technologies. Tissues were counterstained with DAPI (Invitrogen). Images were acquired on a Nikon Eclipse Ni microscope using NIS Elements software, and 5–10 fields were analyzed per tumor.

Goel S., DeCristo M.J., Watt A.C., BrinJones H., Sceneay J., Li B.B., Khan N., Ubellacker J.M., Xie S., Metzger-Filho O., Hoog J., Ellis M.J., Ma C., Ramm S., Krop I.E., Winer E.P., Roberts T.M., Kim H.J., McAllister S.S, & Zhao J.J. (2017). CDK4/6 inhibition triggers anti-tumor immunity. Nature, 548(7668), 471-475.

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Multiplex Immunostaining for Tumor Analysis

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Immunohistochemical Analysis of Tumor-Infiltrating Immune Cells

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Tumour tissues were carefully isolated and fixed in 4% buffered formalin phosphate solution (Fischer Scientific) for 24 h, followed by 70% ethanol solution overnight. The tissues were embedded in paraffin, and 5-μm tissue slices were prepared. For the immunostaining studies, tumour slides were pre-blocked with a blocking solution containing 5% BSA and 10% control normal serum and then stained with primary antibodies including CD4 (clone 4SM95, eBioscience, #14-9766-82, 1:50), CD8 (clone 4SM15, eBioscience, #100702, 1:50), F4/80 (clone CI:A3-1, Abcam, #6640, 1:100), Iba-1 (pAb, Abcam, #ab5076, 1:1000), CD11c (clone N418, eBioscience, #14-0114-82, 1:100), pIRF3 S396 (clone D6O1M, Cell Signaling, #29047, 1:50), PD1 (clone D7D5W, Cell Signaling, #84651, 1:200), and PDL1 (clone D5V3B, Cell Signaling, #64988, 1:200) at 4 °C overnight. After washing, the appropriate fluorescence-labelled secondary antibodies (eBioscience) were added for 2 h at room temperature in the dark. Cell nuclei were counterstained with DAPI and the samples were mounted with an antifade mounting medium (ThermoFisher) before imaging. Fluorescence images were obtained with an Olympus Fluoview FV3000 laser scanning microscope and were further analysed with Image J software.

Lu Y., Huntoon K., Lee D., Wang Y., Ha J., Qie Y., Li X., Schrank B.R., Dong S., Gallup T.D., Kang M., Zhao H., An Y., Yang Z., Li J., Kim B.Y, & Jiang W. (2022). Immunological conversion of solid tumours using a bispecific nanobioconjugate for cancer immunotherapy. Nature nanotechnology, 17(12), 1332-1341.

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Immunohistochemical Analysis of Breast and Brain Tissues

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The brain and mammary tissues were snap-frozen in liquid nitrogen in optimal cutting temperature for IHC. Other tissues were fixed in 10% neutral buffered formalin and embedded in paraffin. A total of 4 mmol/L sections were stained with antibodies specific for CD8 (clone 4SM15, eBioscience) and Caspase 3 (9661, Cell Signaling Technology) and were performed as described previously (3, 15) . Herceptin (Roche) was biotinylated using the EZ-Link NHS-Biotin (Thermo Fisher Scientific) according to the manufacturer's instructions. A VECTASTAIN ABC kit was used in combination with DAB (Vector Laboratories) to visualize the cells expressing Her2 antigen. All the slides were counterstained with hematoxylin. Microscopic images were taken using the Olympus VS120 microscope (Olympus) and assembled using the software Adobe Photoshop and Illustrator.

Ali A.I., Wang M., von Scheidt B., Dominguez P.M., Harrison A.J., Tantalo D.G.M., Kang J., Oliver A.J., Chan J.D., Du X., Bai Y., Lee B., Johnstone R.W., Darcy P.K., Kershaw M.H, & Slaney C.Y. (2021). A Histone Deacetylase Inhibitor, Panobinostat, Enhances Chimeric Antigen Receptor T-cell Antitumor Effect Against Pancreatic Cancer. Clinical cancer research : an official journal of the American Association for Cancer Research, 27(22).

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Quantitative analysis of infiltrated immune cells in arthritis

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To detect infiltrated immune cells, paw sections (3.5 µm) were fixed with acetone and denatured with 100, 95, and 75% ethanol for 15 s separately. For antigen retrieval, sections were heated in Tris-EDTA retrieval buffer (10 mM Tris Base, 1 mM EDTA solution, 0.05% Tween 20, pH 9.0) in a water bath at 98°C for 20 min. After cooling down at RT for 30 min, sections were incubated with antibodies against F4/80 (clone CI:A3-1; Abcam), GR-1 (clone RB6-8C5; R&D), CD4 (clone 4SM95; Thermo Fisher), CD8 (clone 4SM15; Thermo Fisher), and CD19 (clone 6OMP31; Thermo Fisher) at 4°C overnight for detecting macrophages, neutrophils, CD4+ T cells, CD8+ T cells, and B cells respectively. Next, sections were washed with PBS and incubated with anti-Rat IgG (Alexa Fluor 488 conjugated; Thermo Fisher) secondary antibody. Cell nucleus was counterstained with Hoechst 33342 (Thermo Fisher), and sections were visualized using a fluorescence microscope (Olympus BX61). Quantification of fluorescence intensity was performed by count function in the software of CellSens dimension (Olympus). Briefly, five different regions in arthritis paw from DTHA mice and normal paw from untreated mice with strongest fluorescence intensity were selected for each sample. Then the average value of fluorescence intensity was calculated from these five regions and used for the next statistical analysis.

Li G., Kolan S.S., Guo S., Marciniak K., Kolan P., Malachin G., Grimolizzi F., Haraldsen G, & Skålhegg B.S. (2021). Activated, Pro-Inflammatory Th1, Th17, and Memory CD4+ T Cells and B Cells Are Involved in Delayed-Type Hypersensitivity Arthritis (DTHA) Inflammation and Paw Swelling in Mice. Frontiers in Immunology, 12, 689057.

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Comprehensive Antibody Profiling for Cell Characterization

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The antibodies used in the study are summarized as follows: CD4 (clone 4SM95; number 14-9766-82), CD8 (clone 4SM15; number 14-0808-82), CD49b/Integrin Subunit Alpha 2 (ITGA2) (clone DX5; number 14-5971-85), forkhead box P3 (FOXP3) (clone FJK-16s; number 14-5773-82), inducible nitric oxide synthase/nitric oxide synthase 2 (number PA1-036), arginase 1 (ARG1; number PA5-29645), pan-cytokeratin (number PA1-27114), CD68 (clone FA-11; number 14-0681-82), and F4/80 (clone BM8; number 14-4801-82) were purchased from ThermoFisher Scientific (Waltham, MA). TP53 (clone POE316A/E9; number MABE1808) and tubulin (number T9026) were from MilliporeSigma (Burlington, MA). HPV16 E6 (number orb10837) for Western blot analysis and HPV16 E7 (number orb10573) for immunofluorescence were from Biorbyt LLC (St. Louis, MO). HPV16 E6 (clone C1P5; number NB100-2729) for immunofluorescence, HPV16 E7 (clone TVG 701Y; number NB100-2768) for Western blot analysis, and bromodeoxyuridine (BrdU; clone BU1/75; number NB500169) were from Novus Biologicals (Centennial, CO).

Li M., McGhee E.M., Shinno L., Lee K, & Lin Y.L. (2021). Exposure to Microbial Metabolite Butyrate Prolongs the Survival Time and Changes the Growth Pattern of Human Papillomavirus 16 E6/E7-Immortalized Keratinocytes in Vivo. The American Journal of Pathology, 191(10), 1822-1836.

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Multiplex Immunohistochemistry for Tumor Immune Profiling

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After harvesting, tumors were immediately fixed overnight in 10% neutral-buffered formalin. Fixed tumors were dehydrated using an ethanol series, embedded in paraffin, and sections were cut at a thickness of 5 μm. Full-section slides of tumor tissues were stained using Opal multiplex 6-plex kits, according to the manufacturer’s protocol (Akoya), for DAPI, Epcam (polyclonal; Abcam, 1:100 dilution), CD3 (clone SP7; Spring Biosciences; 1:100 dilution), CD8 (clone 4SM15; Thermo Fisher; 1:500), CD4 (clone 4SM95; eBioscience, 1:50), Foxp3 (polyclonal; Thermo Fisher, 1:500), and Granzyme B (polyclonal; Abcam, 1:200). Single color controls and an unstained slide were also included for proper spectral un-mixing.

Newton J.M., Hanoteau A., Liu H.C., Gaspero A., Parikh F., Gartrell-Corrado R.D., Hart T.D., Laoui D., Van Ginderachter J.A., Dharmaraj N., Spanos W.C., Saenger Y., Young S, & Sikora A.G. (2019). Immune microenvironment modulation unmasks therapeutic benefit of radiotherapy and checkpoint inhibition. Journal for Immunotherapy of Cancer, 7, 216.

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Multiplex Immunofluorescence Staining and Analysis

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Sectioning and Staining: After harvesting, tumors were immediately fixed overnight in 10% neutral-buffered formalin. Fixed tumors were embedded in paraffin and sections were cut at a thickness of 5 μm. Full-section slides of tumor tissues were stained using Opal multiplex 6-plex kits, according to the manufacturer’s protocol (PerkinElmer), for DAPI, Epcam (polyclonal; Abcam, 1:100 dilution), CD3 (clone SP7; Spring Biosciences; 1:100 dilution), CD8 (clone 4SM15; ThermoFisher; 1:500), CD4 (clone 4SM95; eBioscience, 1:50), Foxp3 (polyclonal; ThermoFisher, 1:500), and Granzyme B (polyclonal; Abcam, 1:200). Single color controls and an unstained slide were also included.
Multispectral imaging and analysis: Multispectral image capture was done at 20X magnification using Vectra (PerkinElmer, Hopkinton, MA). Images were analyzed using inForm software version 2.4.1 (PerkinElmer) as previously described [31 (link)]. Further details are presented in the Supplementary Materials.

Hanoteau A., Newton J.M., Krupar R., Huang C., Liu H.C., Gaspero A., Gartrell R.D., Saenger Y.M., Hart T.D., Santegoets S.J., Laoui D., Spanos C., Parikh F., Jayaraman P., Zhang B., Van der Burg S.H., Van Ginderachter J.A., Melief C.J, & Sikora A.G. (2019). Tumor microenvironment modulation enhances immunologic benefit of chemoradiotherapy. Journal for Immunotherapy of Cancer, 7, 10.

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