Cd8 t cell negative selection kit

Manufactured by STEMCELL

Sourced in Norway

The CD8+ T cell negative selection kit is a laboratory tool used for the isolation of CD8+ T cells from a mixed cell population. It removes non-CD8+ T cells, leaving the desired CD8+ T cell population. The kit utilizes an antibody-based approach to selectively deplete unwanted cells, providing a simple and efficient method for CD8+ T cell purification.

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Lab products found in correlation

Rpmi 1640, by Thermo Fisher Scientific (1 mentions) L glutamine, by Thermo Fisher Scientific (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Hepes, by Corning (1 mentions) Pen strep, by Corning (1 mentions) Ficoll, by GE Healthcare (1 mentions) Interleukin 2 (il 2), by R&D Systems (1 mentions) Dynabeads human t activator cd3 cd28, by Thermo Fisher Scientific (1 mentions) Celltrace violet, by Thermo Fisher Scientific (1 mentions) Interleukin 2 (il 2), by Thermo Fisher Scientific (1 mentions) Cd4 t cell negative selection kit, by STEMCELL (1 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions) Lyse fix buffer, by BD (1 mentions) Perm buffer 3, by BD (1 mentions) Il 27, by R&D Systems (1 mentions) Ifn β, by PBL Assay Science (1 mentions) Ifn 1, by R&D Systems (1 mentions) Brefeldin a, by Merck Group (1 mentions) Anti cd16 32, by BioXCell (1 mentions) Phorbol 12 myristate 13 acetate pma ionomycin, by Merck Group (1 mentions)

Spelling variants of this product

Negative selection kit (58 citations) Negative selection (55 citations) CD8 negative selection (5 citations)

8 protocols using cd8 t cell negative selection kit

Adoptive Transfer of OT-I CD8+ T Cells

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For adoptive transfers CD8⁺ T cells were enriched from spleens of OT-I mice using the CD8⁺ T cell negative selection kit (Stem Cell, 19853), followed by sorting of naïve CD62L^hi CD44^lo CD8⁺ T cells (BD Aria II). Mixed 1:1 ratio (for co-transfers) 5×10⁴ cells were transferred intravenously into WT CD45.1 mice, 1 day prior to infection. Donor cells were subsequently assessed at indicated time points post-infection in different experiments.
For Lm-Ova InlA^M experiments, mice were food starved for several hours during the day and infected late afternoon by feeding infected bread containing 2×10⁹ (primary) or 2×10¹⁰ (reinfection) colony forming units (CFUs) of Lm-Ova InlA^M.

Biggs J.R., Yang J., Gullberg U., Muchardt C., Yaniv M, & Kraft A.S. (2001). The human brm protein is cleaved during apoptosis: The role of cathepsin G. Proceedings of the National Academy of Sciences of the United States of America, 98(7), 3814-3819.

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Adoptive Transfer of OT-I CD8+ T Cells

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OVA-specific CD8⁺ T cells were obtained from spleen and inguinal and axial lymph nodes of OT-I TCR transgenic C57BL/6 mice (Ly5.2). The CD8⁺ T cell negative selection kit (Stemcell Technologies) was used to isolate a pure population of CD8⁺ T cells (98% CD8⁺ T cells as determined by FACS). OT-I T cells were then cultured in 10-cm plates prebound with anti-CD3 and anti-CD28 antibodies (10 μg/mL each) for 72 h in Roswell Park Memorial Institute (RPMI) medium +10% FBS. On day 22 posttumor cell injection, 2 × 10⁶ OT-I cells were injected i.v. into MC38-OVA−bearing C57BL/6 (Ly5.1) mice. Eight days post–OT-I adoptive transfer (day 30 posttumor cell injection), mice were killed, and tumor and TDLNs (right inguinal and axial lymph nodes) were collected for assessment by flow cytometry (as per SI Appendix).

Kashyap A.S., Schmittnaegel M., Rigamonti N., Pais-Ferreira D., Mueller P., Buchi M., Ooi C.H., Kreuzaler M., Hirschmann P., Guichard A., Rieder N., Bill R., Herting F., Kienast Y., Dirnhofer S., Klein C., Hoves S., Ries C.H., Corse E., De Palma M, & Zippelius A. (2019). Optimized antiangiogenic reprogramming of the tumor microenvironment potentiates CD40 immunotherapy. Proceedings of the National Academy of Sciences of the United States of America, 117(1), 541-551.

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Primary T Cell Cytotoxicity Assay

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Deidentified buffy coats from healthy human donors were obtained from Massachusetts General Hospital. PBMCs were isolated by density-based centrifugation using Ficoll (GE Healthcare). CD8+ T cells were isolated from PBMCs using a CD8+ T cell-negative selection kit (Stemcell). T cells were mixed with Human T-activator CD3/CD28 DynaBeads (Thermo Fisher Scientific) in a 1:1 ratio and maintained in R10 + IL-2 [RPMI 1640 (Thermo Fisher Scientific) supplemented with 10% heat-inactivated FBS (Thermo Fisher Scientific), 1% HEPES (Corning), 1% L-glutamine (Thermo Fisher Scientific), 1% Pen/Strep (Corning) and 50 IU/mL of IL-2 (R&D Systems)] for 7 d prior to use in cell killing assays. DynaBeads were removed by magnetic separation prior to co-incubation of primary T cells with target cells. Target cells were treated with DMSO or 1 µM MEKi for 72 h and were subsequently seeded in a 96-well plate with primary T cells in R10 + IL-2 at an effector to target ratio of 2:1 and incubated with BiTEs for 48 h with n = 3 technical replicates per condition. Cells were assayed with CTG, and percent cytotoxicity was calculated by subtracting the average luminescence signal of the T cell only condition and normalizing to the no BiTE condition. ((X − [T cell only])/([average-no-BiTE] – [T cell only])) × 100.

Stopfer L.E., Rettko N.J., Leddy O., Mesfin J.M., Brown E., Winski S., Bryson B., Wells J.A, & White F.M. (2022). MEK inhibition enhances presentation of targetable MHC-I tumor antigens in mutant melanomas. Proceedings of the National Academy of Sciences of the United States of America, 119(49), e2208900119.

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CD8+ T Cell Proliferation Assay

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CD8⁺ T cells were enriched from spleens of C57BL/6J mice using a CD8⁺ T cell negative selection kit (Stem Cell Technologies #19853) and then isolated through flow sorting. CD4⁺ T_reg cells were enriched using a CD4⁺ T cell negative selection kit (Stem Cell Technologies #19765) and then isolated through flow sorting. CD8⁺ T cells were stained with Cell Trace Violet (Thermofisher Scientific #C34557) and then 2.5x10⁴ cells were plated per well in RPMI (Cytiva #SH30255.02) in a 96 well plate with 7.5x10⁴ CD3/CD28 activation beads (Miltenyi Biotec #130-095-925), 10% fetal bovine serum (FBS) (R&D Systems #S11150), and 30 IU/mL IL2 (PeproTech #212-12). Varying ratios of T_reg cells were incubated with CD8⁺ T cells for 72 h and then CD8 T cell proliferation was measured by flow cytometry analysis of Cell Trace Violet dilution.⁴² (link)

Chaudhuri S.M., Weinberg S.E., Wang D., Yalom L.K., Montauti E., Iyer R., Tang A.Y., Torres Acosta M.A., Shen J., Mani N.L., Wang S., Liu K., Lu W., Bui T.M., Manzanares L.D., Dehghani Z., Wai C.M., Gao B., Wei J., Yue F., Cui W., Singer B.D., Sumagin R., Zhang Y, & Fang D. (2024). Mediator complex subunit 1 architects a tumorigenic Treg cell program independent of inflammation. Cell Reports Medicine, 5(3), 101441.

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Assessing STAT1 Phosphorylation and IL-27R Expression

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For a flow cytometric analysis of STAT1 phosphorylation, cells were treated with IFN-I (5,000 U/ml) or IL-27 (50 ng/ml; R&D Systems) for 30 min and then fixed with Lyse/Fix Buffer (BD Biosciences) for 12 min at 37°C. Surface markers were stained after fixation. Cells were then permeabilized with Perm Buffer III (BD Biosciences) for 30 min on ice, washed, and stained for phosphorylated STAT1 (BD Biosciences). For analysis of IFN-I effect on IL-27R expression, mouse CD8⁺ T cells from spleen were purified with CD8⁺ T cell negative selection kit (Stemcell Technologies) and stimulated with plate-coated anti-CD3 and anti-CD28 (BioLegend) in the absence or presence of 5,000 U/ml IFNβ (PBL Assay Science) for 2 d. Il27RA and GP130 expression were examined by flow cytometry. For RNA-seq analysis, splenic CXCR5⁺ CD8⁺ and CXCR5⁻ CD8⁺ T cells were purified by flow cytometry, plated at a concentration of 10⁶ cell/ml, and treated with growth medium (RPMI 1640 containing 10% FBS and supplemented with nonessential amino acids, sodium pyruvate, Hepes, l-glutamine, 2-mercaptoethanol, and penicillin-streptomycin [Gibco]) with or without recombinant IL-27 (20 ng/ml; R&D Systems) or recombinant IFNβ (1,000 U/ml, carrier-free; PBL Assay Science) for 12 h at 37°C.

Huang Z., Zak J., Pratumchai I., Shaabani N., Vartabedian V.F., Nguyen N., Wu T., Xiao C, & Teijaro J.R. (2019). IL-27 promotes the expansion of self-renewing CD8+ T cells in persistent viral infection. The Journal of Experimental Medicine, 216(8), 1791-1808.

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Murine Immune Cell Profiling for Malaria

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Male BALB/c mice (6–8 weeks old) used for this study were maintained under pathogen-free condition at institutional animal house facility. Anti-mouse antibodies Alexa Fluor 700-CD8 (Clone 53-6.7), APC-cy7-CD19 (Clone 1D3), APC-cy7-CD45R/B220 (Clone RA3-6B2), were procured from BD Biosciences (San Jose, CA, USA), FITC-CD4 (clone GK 1.5), FITC CD278 (7E.17G9), APC-CD278 (Clone-C398.4A), PerCP-Cy 5.5-CD4 (clone RM4-5), PE-Cy7-IFN-γ (clone XMG 1.2), PE-T-bet (clone eBio 4B10), Alexa Fluor 700-CD8 (53-6.7) were from eBioscience (San Diego, CA, USA), Brilliant violet 605 TCR Vb (clone H57-597), APC-CD278 (clone C398.4A), Anti-CD49b (clone DX5) were from BioLegend (San Diego, CA, USA). Purified antibodies Anti-mouse CD278 ICOS (clone 7E.17G9), Anti-Rat IgG2b isotype control, and anti-CD16/32 were procured from BioXcell (West Lebanon, USA). Dynabeads untouched mouse CD4⁺ T cell isolation kit was procured from Life Technologies AS (Oslo, Norway) and CD8⁺ T cell negative selection kit was from Stem cell technologies (Vancouver, BC, Canada). Phorbol 12-myristate 13-acetate (PMA), Ionomycin, Brefeldin A was procured from Sigma-Aldrich (St. Louis, MO, USA). Malarial parasite P. berghei ANKA (MRA-671, MR4, ATCC, Manassas, VA, USA) was obtained from MR4 repository, ATCC, Manassas, VA, USA.

Jogdand G.M., Sengupta S., Bhattacharya G., Singh S.K., Barik P.K, & Devadas S. (2018). Inducible Costimulator Expressing T Cells Promote Parasitic Growth During Blood Stage Plasmodium berghei ANKA Infection. Frontiers in Immunology, 9, 1041.

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Generating Induced CD103+ Dendritic Cells

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Induced iCD103⁺ DCs were generated as published in Mayer et al.⁵⁵ (link) Briefly, bone marrow cells were isolated from the long bones of C57BL/6 mice and plated at 15.10⁶ cells in 10 mL of complete Lutz media (RPMI supplemented with 10% FBS, 1% Pen/Strep, 25 mM HEPES, and 50 μM β-mercaptoethanol) supplemented with 200 ng/mL Flt3L (made in-house) and 5 ng/mL GM-CSF (Peprotech). On day 5 after plating, 5 mL of complete Lutz media (without added cytokines) was added to each dish. Cells were then harvested on day 9 and replated at 3.10⁶ cells in 10mL of complete Lutz media with added 200 ng/mL Flt3L and 5 ng/mL GM-CSF. Finally, iCD103⁺ DCs were harvested for use on day 16. For the presentation assay, iCD103⁺ DCs were plated (2.10⁴ cells per well in a 96 well plate) with antigen at the given concentration with 5 ng/mL LPS and incubated at 37°C for 9 h. During that time, OT-I cells were isolated using CD8 T cell negative selection kits (STEMCELL Technologies) and labeled using 5 μM CFSE. After the incubation, the antigen loaded DCs were then washed in complete Lutz media before 1.10⁵ CFSE labeled T cells were added to each well. Cells were then incubated for 3 days before staining and analysis by flow cytometry on a BD Fortessa.

Goldberger Z., Hauert S., Chang K., Kurtanich T., Alpar A.T., Repond G., Wang Y., Gomes S., Krishnakumar R., Siddarth P., Swartz M.A., Hubbell J.A, & Briquez P.S. (2023). Membrane-localized neoantigens predict the efficacy of cancer immunotherapy. Cell Reports Medicine, 4(8), 101145.

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Assessing T Cell Proliferation from MN Matrices

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To see the effect of the MN matrices on T cell proliferation and expansion, CD11c+ DCs isolated as previously mentioned were treated with the MN solution (0.0001-100µg), LPS, and PBS. Soluble SIINFEKL (5µg/mL) was also pulsed into the wells along with the MN substrates and controls. After 48h, T cells isolated from OT-1 mice using CD8+ T cell negative selection kits (StemCell Technologies, 19852) were stained with cell proliferation dye eFluor 670 (0.5µM/well) during a 5 min incubation at room temperature. T cells were then co-cultured with each DC sample by adding 3x 10⁵ T cells per well (making the ratio 1:3 DC to T cells). T cell proliferation was determined by the mean fluorescence intensity of the eFluor 670 signal and compared with the positive and negative controls.

Shah S.A., Oakes R.S., Kapnick S.M, & Jewell C.M. (2022). Mapping the Mechanical and Immunological Profiles of Polymeric Microneedles to Enable Vaccine and Immunotherapy Applications. Frontiers in Immunology, 13, 843355.

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