Cd8 clone 3b5

Manufactured by Thermo Fisher Scientific

The CD8 (clone 3B5) is a monoclonal antibody that binds to the CD8 molecule expressed on the surface of certain T cells. It is a laboratory tool used for the identification and analysis of CD8-positive cells in biological samples.

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Lab products found in correlation

Rnalater, by Thermo Fisher Scientific (2 mentions) Fixable violet dead cell stain kit, by Thermo Fisher Scientific (1 mentions) Pd 1 clone j105, by Thermo Fisher Scientific (1 mentions) Cd3 clone sp34 2, by BD (1 mentions) Cd4 clone l200, by BD (1 mentions) Hla dr clone l243, by BD (1 mentions) Pd 1 clone eh12.2h7, by BioLegend (1 mentions) Foxp3 clone pch101, by Thermo Fisher Scientific (1 mentions) Ki67 clone b56, by BD (1 mentions) Cd28 clone cd28, by Beckman Coulter (1 mentions) Cd95 clone dx2, by BioLegend (1 mentions) Cd127 clone hil 7r m21, by BD (1 mentions) Lsr 2 flow cytometer, by BD (1 mentions) Live dead fixable violet dead cell stain, by Thermo Fisher Scientific (1 mentions) Cd4 clone sk3, by Thermo Fisher Scientific (1 mentions) Cd154, by BD (1 mentions) Cd154 clone trap1, by BD (1 mentions) Cd137, by BD (1 mentions) Cd14 clone m5e2, by BioLegend (1 mentions) Cd19 clone hib19, by BioLegend (1 mentions)

3 protocols using cd8 clone 3b5

Phenotypic Characterization of TIL Infusion

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A small fraction of TIL infusion products was cryopreserved for subsequent phenotypic characterization by flow cytometry. In brief, after thawing, cells were rested for 24 hours, and then stained using antibodies directed against: CD3 (clone SK7), CCR7 (clone 150503), CD28 (clone CD28.2), CD62L (clone SK11) (all BD Biosciences), CD4 (clone S3.5), CD8 (clone 3B5), CD45RA (clone MEM-56), CD27 (clone CLB-27/1) (all Invitrogen) and PD-1 (clone J105, eBioscience). Fixable Violet Dead Cell Stain Kit (Invitrogen) was used to exclude dead cells. Cells were analyzed on a LSR2 SORP flow cytometer (BD Biosciences) and data was processed using Flowjo_V10 software.

van den Berg J.H., Heemskerk B., van Rooij N., Gomez-Eerland R., Michels S., van Zon M., de Boer R., Bakker N.A., Jorritsma-Smit A., van Buuren M.M., Kvistborg P., Spits H., Schotte R., Mallo H., Karger M., van der Hage J.A., Wouters M.W., Pronk L.M., Geukes Foppen M.H., Blank C.U., Beijnen J.H., Nuijen B., Schumacher T.N, & Haanen J.B. (2020). Tumor infiltrating lymphocytes (TIL) therapy in metastatic melanoma: boosting of neoantigen-specific T cell reactivity and long-term follow-up. Journal for Immunotherapy of Cancer, 8(2), e000848.

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Multiparameter Phenotypic Characterization of PBMCs

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Thawed PBMCs were surface stained with Aqua amine reactive viability dye (Invitrogen) and mAbs to CD45 (clone D058–1283, BD Biosciences), CD3 (clone SP34–2, BD Biosciences), CD4 (clone L200, BD Biosciences), CD8 (clone 3B5, Invitrogen), CD95 (clone DX2, BioLegend), CD28 (clone CD28.2, Beckman), CD127 (clone hIL-7R-M21, BD Biosciences), PD-1 (clone EH12.2H7, BioLegend), and HLA-DR (clone L243, BD Biosciences) for 20 min at room temperature, fixed, and then permeabilized with FoxP3 Fix/Perm Buffers (eBioscience) per the manufacturer’s instructions. Permeabilized cells were then stained intracellularly for Ki67 (clone B56, BD Biosciences) and FoxP3 (clone PCH101, eBioscience) for 30 min at 4°C, washed in FoxP3 Perm/Wash buffer, fixed in 0.5% paraformaldehyde and analyzed by flow cytometry on a BD LSRII flow cytometer (BD Biosciences) and analyzed using Flowjo Software (Tree Star Inc).

Swainson L.A., Ahn H., Pajanirassa P., Khetarpal V., Deleage C., Estes J.D., Hunt P.W., Munoz-Sanjuan I, & McCune J.M. (2019). Kynurenine 3-monooxygenase inhibition during acute SIV infection lowers PD-1 expression and improves post-cART CD4+ T cell counts and body weight: KMO inhibition improves clinical outcome in SIV infection. Journal of immunology (Baltimore, Md. : 1950), 203(4), 899-910.

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Neoantigen-reactive CD4+ T-cell Isolation

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Cryopreserved PBMC were thawed and cultured at 2×10⁶ cells/mL in a 24-well plate with anti-CD40 at 500 ng/mL and each of the five neoantigen peptides as well as a control HIV NEF (RYPLTFGWCF) peptide at 1 µg/mL overnight at 37°C. After 16 hours, cells were stained for with LIVE/DEAD Fixable Violet Dead Cell Stain (Invitrogen), followed by lineage markers CD14 (clone M5E2, Biolegend), CD19 (clone HIB19, Biolegend), CD4 (clone SK3, eBioscience), CD8 (clone 3B5, Invitrogen) and activation-induced markers CD69 (clone L78, BD Biosciences), CD137 (clone 4-1BB, Biolegend) and CD154 (clone TRAP1, BD Bioscience). Single neoantigen-reactive CD4 cells were sorted into RNAlater (Thermo Fisher) based on coexpression of CD69, CD137 and CD154 on a BD FACSAria II (BD Biosciences).

Church C., Pulliam T., Longino N., Park S.Y., Smythe K.S., Makarov V., Riaz N., Jing L., Amezquita R., Campbell J.S., Gottardo R., Pierce R.H., Choi J., Chan T.A., Koelle D.M, & Nghiem P. (2022). Transcriptional and functional analyses of neoantigen-specific CD4 T cells during a profound response to anti-PD-L1 in metastatic Merkel cell carcinoma. Journal for Immunotherapy of Cancer, 10(9), e005328.

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