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Cd8 negative isolation kits

Manufactured by Thermo Fisher Scientific
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The CD8 negative isolation kits are designed to isolate untouched CD8-positive T cells from human peripheral blood mononuclear cells (PBMCs) or other samples. The kits utilize a negative selection approach, where CD8-positive cells are magnetically labeled and removed, leaving the unlabeled CD8-negative cells available for further downstream applications.

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Lab products found in correlation

Mouse ifnγ elisa ready set go assay, by Thermo Fisher Scientific (2 mentions) Permwash, by BioLegend (1 mentions) Perm wash, by BD (1 mentions) Golgistop, by BD (1 mentions) Cytofix cytoperm buffer, by BD (1 mentions) Ifnγ bv421, by BioLegend (1 mentions) Mouse ifnγ elisa ready set go, by Thermo Fisher Scientific (1 mentions) Aim 5 medium, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

Negative isolation kits (6 citations) Dynal mouse CD4 or CD8 negative isolation kits (2 citations)

7 protocols using cd8 negative isolation kits

1

Isolated CD8+ T Cell Stimulation

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Isolated liver infiltrating lymphocytes and splenocytes were enriched for CD8 cells using CD8 negative isolation kits (Life Technologies, Frederick, MD, USA) according to manufacturer's protocol. Irradiated Panc02 tumor cells were added to isolated CD8+ T cells at a ratio of 5:1 (2x105 CD8+ T cells with 4x104 Panc02 tumor cells) and incubated for 18 hours at 37°C. Mouse IFNγ ELISA Ready-SET-Go assay was conducted per manufacturer protocol (eBioscience).
Soares K.C., Rucki A.A., Wu A.A., Olino K., Xiao Q., Chai Y., Wamwea A., Bigelow E., Lutz E., Liu L., Yao S., Anders R.A., Laheru D., Wolfgang C.L., Edil B.H., Schulick R.D., Jaffee E.M, & Zheng L. (2015). PD-1/PD-L1 blockade together with vaccine therapy facilitates effector T cell infiltration into pancreatic tumors. Journal of immunotherapy (Hagerstown, Md. : 1997), 38(1), 1-11.
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2

Characterization of CD8+ T-cell responses

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Isolated liver infiltrating lymphocytes were enriched for CD8+ cells using CD8-negative isolation kits (LifeTechnologies) according to the protocol provided by the manufacturer. Peptide-stimulated Kb and Db cells were added to isolated CD8+ T cells at a ratio of 1:1 (2 × 105 cells of each respective cell line) and incubated for 18 h at 37 °C. Mouse IFNγ Enzyme-Linked Immunosorbent Assay (ELISA) Ready-Set-Go assay was conducted per the manufacturer’s protocol (eBioscience). Mouse IFNγ Enzyme-Linked ImmunoSpot (ELISPOT) assay was conducted per the manufacturer’s protocol (Abcam).
Kim V.M., Blair A.B., Lauer P., Foley K., Che X., Soares K., Xia T., Muth S.T., Kleponis J., Armstrong T.D., Wolfgang C.L., Jaffee E.M., Brockstedt D, & Zheng L. (2019). Anti-pancreatic tumor efficacy of a Listeria-based, Annexin A2-targeting immunotherapy in combination with anti-PD-1 antibodies. Journal for Immunotherapy of Cancer, 7, 132.
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3

Enrichment and Stimulation of CD8+ T Cells

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Isolated liver infiltrating lymphocytes and splenocytes were enriched for CD8 cells using CD8 negative isolation kits (Life Technologies, Frederick, MD, USA) according to manufacturer's protocol. CD3:CD28 stimulation beads (Life Technologies, Frederick, MD, USA) were added to isolated CD8+ T cells and incubated for 12 hours at 37°C in 5% CO2 according manufacturer's protocol. Golgistop (1:1000; BD Biosciences) was added and incubated for 5 hours at 37°C in 5% CO2. After removing the beads according to manufacturer's protocol and washing the cells twice with flow buffer, cells were stained with CD8, CD3 and live dead Near-IR stain according to the above protocol. The cells were then washed twice, suspended in cytofix/cytoperm buffer (BD Biosciences), incubated at 4°C for 30 minutes and then washed with Permwash (BD Biosciences). IFNγ-BV421 (Biolegend) antibody was added in Permwash and incubated at 4°C for 20 minutes. Flow cytometry assays were completed on an LSR II flow cytometer.
Soares K.C., Rucki A.A., Wu A.A., Olino K., Xiao Q., Chai Y., Wamwea A., Bigelow E., Lutz E., Liu L., Yao S., Anders R.A., Laheru D., Wolfgang C.L., Edil B.H., Schulick R.D., Jaffee E.M, & Zheng L. (2015). PD-1/PD-L1 blockade together with vaccine therapy facilitates effector T cell infiltration into pancreatic tumors. Journal of immunotherapy (Hagerstown, Md. : 1997), 38(1), 1-11.
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4

CD8+ T Cell Activation Assay

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CD8+ T cells from liver infiltrating lymphocytes and splenocytes were isolated using CD8 negative isolation kits (Life Technologies) according to the manufacturer's protocol. Irradiated Panc02 tumor cells were added to isolated CD8 T cells at a ratio of 5:1 (2 × 105 CD8+ T cells combined with 4 × 104 Panc02 tumor cells) and were subsequently incubated for 18 hours in 5% CO2 at 37°C. The ELISA assay was then conducted using mouse IFNγ ELISA Ready-SET-Go assay per the manufacturer's protocol (eBioscience).
Soares K.C., Rucki A.A., Kim V., Foley K., Solt S., Wolfgang C.L., Jaffee E.M, & Zheng L. (2015). TGF-β blockade depletes T regulatory cells from metastatic pancreatic tumors in a vaccine dependent manner. Oncotarget, 6(40), 43005-43015.
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5

Antitumor CD8+ T cell assay

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CD8-negative isolation kits (Life Technologies) were used to isolate CD8+ T cells from the liver and spleen of mice that underwent the hemispleen procedure for the metastatic tumor model. The isolated CD8+ cells from the same treatment group were pooled and cocultured with irradiated (at 50 Gy) autologous KPC tumor cells at a ratio of 5:1 (2 × 105 CD8+ T cells: 4 × 104 irradiated KPC tumor cells). The coculture was incubated for 18 h in AIM V  medium (Thermo Fisher Scientific) at 37°C. Mouse IFN-γ ELISA Ready-Set-Go (eBioscience) was then conducted with the supernatant per manufacturer’s protocol.
Wang J., Saung M.T., Li K., Fu J., Fujiwara K., Niu N., Muth S., Wang J., Xu Y., Rozich N., Zlomke H., Chen S., Espinoza B., Henderson M., Funes V., Herbst B., Ding D., Twyman-Saint Victor C., Zhao Q., Narang A., He J, & Zheng L. (2022). CCR2/CCR5 inhibitor permits the radiation-induced effector T cell infiltration in pancreatic adenocarcinoma. The Journal of Experimental Medicine, 219(5), e20211631.
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6

Priming Mice for T Cell Assay

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Mice were primed intranasally with the following viruses at indicated doses: live PR8 (0.001 HAU), BPL-inactivated PR8 (3800 HAU). Groups of 3 mice were primed per condition. Mouse spleens were harvested 10–12 days post-priming, pooled, and CD4+ or CD8+ T cells were purified using Dynal CD4 or CD8 Negative Isolation Kits according to manufacturer’s instructions (Invitrogen). BMDC or MuIFN-γ-activated DC2.4 were used as APC and co-cultured overnight with purified CD4+ T cells (105 per well in all cases) in the presence of indicated peptides and screened for production of IFN-γ as a readout of T-cell activation. Overlapping peptide libraries were screened in triplicate as either matrixed peptide pools (containing 8–12 peptides/well)41 (link) or individual peptides at a final concentration of 10 μg/ml. For all ELISpot assays, baselines were calculated as 2s.d. above background.
Miller M.A., Ganesan A.P., Luckashenak N., Mendonca M, & Eisenlohr L.C. (2015). Endogenous antigen processing drives the primary CD4+ T cell response to influenza. Nature medicine, 21(10), 1216-1222.
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7

Priming Mice for T Cell Assay

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Mice were primed intranasally with the following viruses at indicated doses: live PR8 (0.001 HAU), BPL-inactivated PR8 (3800 HAU). Groups of 3 mice were primed per condition. Mouse spleens were harvested 10–12 days post-priming, pooled, and CD4+ or CD8+ T cells were purified using Dynal CD4 or CD8 Negative Isolation Kits according to manufacturer’s instructions (Invitrogen). BMDC or MuIFN-γ-activated DC2.4 were used as APC and co-cultured overnight with purified CD4+ T cells (105 per well in all cases) in the presence of indicated peptides and screened for production of IFN-γ as a readout of T-cell activation. Overlapping peptide libraries were screened in triplicate as either matrixed peptide pools (containing 8–12 peptides/well)41 (link) or individual peptides at a final concentration of 10 μg/ml. For all ELISpot assays, baselines were calculated as 2s.d. above background.
Miller M.A., Ganesan A.P., Luckashenak N., Mendonca M, & Eisenlohr L.C. (2015). Endogenous antigen processing drives the primary CD4+ T cell response to influenza. Nature medicine, 21(10), 1216-1222.
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