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Na ve cd8 t cell isolation kit

Manufactured by Miltenyi Biotec
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The Naïve CD8+ T cell isolation kit is a laboratory product designed to isolate naïve CD8+ T cells from biological samples. The kit utilizes a magnetic bead-based separation method to enrich for the desired cell population.

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Lab products found in correlation

Anti cd3, by Thermo Fisher Scientific (3 mentions) Anti cd28, by Thermo Fisher Scientific (3 mentions) Interleukin 2 (il 2), by Thermo Fisher Scientific (2 mentions) Sodium pyruvate, by Thermo Fisher Scientific (2 mentions) Hepes, by Thermo Fisher Scientific (2 mentions) Cd11c microbeads, by Miltenyi Biotec (1 mentions) Interleukin 7 (il 7), by R&D Systems (1 mentions) Dynabead, by Thermo Fisher Scientific (1 mentions) L glutamine, by Corning (1 mentions) Dulbecco s modified eagle s medium, by Corning (1 mentions) Dmrie c reagent, by Thermo Fisher Scientific (1 mentions) Transcriptaid t7 high yield transcription kit, by Thermo Fisher Scientific (1 mentions) Foetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Huh 7.5 cells, by American Type Culture Collection (1 mentions) Soluble anti cd28, by Thermo Fisher Scientific (1 mentions) Tgf β, by R&D Systems (1 mentions) Ifn β, by R&D Systems (1 mentions) Anti cd28 clone 37, by BioXCell (1 mentions) Anti cd3 clone 145 2c11, by BioXCell (1 mentions) Rpmi 1640, by Thermo Fisher Scientific (1 mentions)

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CD8+ T cells (21 citations) Isolation kits (10 citations) Cell isolation kits (5 citations) CD8+ isolation kits (2 citations)

16 protocols using na ve cd8 t cell isolation kit

1

Isolation and Enrichment of Myeloid, OTI, and OTII Cells

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For neonatal PP a dissection microscope was used. PP were excised, counted and digested using 0.2 mg/mL Liberase TH/DNAse I for 45 min at 37°C and then dissociated by vigorous pipetting. Myeloid cells were positively enriched using CD11c MicroBeads (Miltenyi). OTI and OTII cells were isolated from lymph nodes and digested using 0.2 mg/mL Liberase TH/DNAse I for 45 min at 37°C and then dissociated by vigorous pipetting. OTI cells were then further enriched using a naïve CD8+ T cell isolation kit (Miltenyi).
Torow N., Li R., Hitch T.C., Mingels C., Al Bounny S., van Best N., Stange E.L., Simons B., Maié T., Rüttger L., Gubbi N.M., Abbott D.A., Benabid A., Gadermayr M., Runge S., Treichel N., Merhof D., Rosshart S.P., Jehmlich N., Hand T.W., von Bergen M., Heymann F., Pabst O., Clavel T., Tacke F., Lelouard H., Costa I.G, & Hornef M.W. (2023). M cell maturation and cDC activation determine the onset of adaptive immune priming in the neonatal Peyer’s patch. Immunity, 56(6), 1220-1238.e7.
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2

Isolation and Activation of Naive CD8+ T Cells

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T cell isolation was performed using the naïve CD8+ T cell isolation kit (Miltenyi Biotec). Spleens and mesenteric lymph nodes were isolated from donor mice and processed according to the manufacturer’s protocol. Naïve CD8+ T cells were then either used directly or activated with anti-CD3 (2 μg/ml) and anti-CD28 (1 μg/ml) (both eBioscience) for 16 h and expanded for 6 days in IL-2 (15 ng/ml, in-house produced).
Haas L., Elewaut A., Gerard C.L., Umkehrer C., Leiendecker L., Pedersen M., Krecioch I., Hoffmann D., Novatchkova M., Kuttke M., Neumann T., de Silva I.P., Witthock H., Cuendet M.A., Carotta S., Harrington K.J., Zuber J., Scolyer R.A., Long G.V., Wilmott J.S., Michielin O., Vanharanta S., Wiesner T, & Obenauf A.C. (2021). Acquired resistance to anti-MAPK targeted therapy confers an immune-evasive tumour microenvironment and cross-resistance to immunotherapy in melanoma. Nature cancer, 2(7), 693-708.
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3

Naïve CD8+ T Cell Isolation and Expansion

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Naïve CD8+ T cells were isolated in a two-step procedure consisting of negative selection followed by positive selection from leukapheresis products using the naïve CD8+ T cell isolation kit (Miltenyi Biotec) and were cultured as reported previously (9 (link)). Briefly, cells were seeded at a concentration of 2.5 x 105 cells/mL in complete RPMI formulated as previously described (9 (link)) and stimulated with α-CD3/28 coated beads (Dynabeads, Thermo Fisher) at a 1:1 ratio with 100 IU/mL recombinant human IL-2 (Teceleukin, NIH). Media and IL-2 were added assuming consumption every 2-3 days, with 5 ng/mL IL-7 (R&D systems) added every 2-3 days after day 5 of culture.
Peters L.D., Yeh W.I., Arnoletti J.M., Brown M.E., Posgai A.L., Mathews C.E, & Brusko T.M. (2023). Modeling cell-mediated immunity in human type 1 diabetes by engineering autoreactive CD8+ T cells. Frontiers in Immunology, 14, 1142648.
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4

Naïve CD8+ T Cell Isolation and Co-Culture

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Naïve CD8+ T cells from the healthy controls and patients were isolated with a Naïve CD8+ T Cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). The cells were washed with RPMI 1640 and then used for co-culture, stimulation, and a flow-cytometric analysis. Naïve CD8+ T cells, PBMCs, and/or Huh 7.5 cells (American Type Culture Collection, Manassas, VA, USA) were co-cultured with Dulbecco’s modified Eagle’s medium (Mediatech Inc., Manassas, VA, USA) containing 2% foetal bovine serum (Life Technologies, Grand Island, NY, USA), 0.05 mM β-mercaptoethanol, 2 M l-glutamine (Mediatech Inc.), and 100 µg/ml penicillin–streptomycin. JFH/Huh 7.5 cells were produced by transfecting cells with the FL-J6/JFH chimeric strain of HCV. Briefly, HCV FL-J6/JFH mRNA was transcribed with a TranscriptAid T7 High Yield Transcription Kit (Fermentas, Vilnius, Lithuania), as previously described.31 (link) Huh7.5 cells were transfected with the transcribed mRNA using DMRIE-C Reagent (Invitrogen). The transfected Huh7.5 cells were collected after 24 h for further experiments. The transfection efficiency was verified with an anti-NS5 antibody (BD Bioscience), as shown in Supplementary Figure 1(a).
Li B.J., He Y., Zhang Y., Guo Y.H., Zhou Y., Zhang P.X., Wang W., Zhao J.R., Li J.G., Zuo W.Z., Fan C, & Jia Z.S. (2017). Interferon-α-induced CD100 on naïve CD8+ T cells enhances antiviral responses to hepatitis C infection through CD72 signal transduction. The Journal of International Medical Research, 45(1), 89-100.
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5

Generation of Tc1/Trm-like Cells from Naive CD8 T Cells

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Naïve CD8 T cells were purified by magnetic selection from healthy donor peripheral blood mononuclear cells [PBMC] using the naïve CD8 T cell isolation kit [Miltenyi Biotec] and were >98% CD8+ and >98% CD45RA+. Naïve CD8 T cells were stimulated with plate-bound anti-CD3 [1 µg/ml], soluble anti-CD28 [1 µg/ml], and IL-2 [5 ng/ml, Peprotech]. Further additions of TGF-β [3 ng/ml, R&D Systems], IFN-β [10 ng/ml, R&D], all-trans retinoic acid [ATRA; 10 nM, Sigma], FICZ [AhR agonist; 100 nM, Tocris Bioscience] were made at the start of the 7-day culture. Cultured cells were washed in PBS, stained for viability and surface or intracellular markers as above. Tc1/Trm-like cells were analysed for cytokine production by re-stimulation with PMA [20 ng/ml] + ionomycin [400 ng/ml] + monensin [3 µM] for 4 h before staining using Foxp3 staining buffer set.
Noble A., Durant L., Hoyles L., Mccartney A.L., Man R., Segal J., Costello S.P., Hendy P., Reddi D., Bouri S., Lim D.N., Pring T., O’Connor M.J., Datt P., Wilson A., Arebi N., Akbar A., Hart A.L., Carding S.R, & Knight S.C. (2019). Deficient Resident Memory T Cell and CD8 T Cell Response to Commensals in Inflammatory Bowel Disease. Journal of Crohn's & Colitis, 14(4), 525-537.
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6

Isolation and Activation of Naive CD8+ T Cells

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T cell isolation was performed using the naïve CD8+ T cell isolation kit (Miltenyi Biotec). Spleens and mesenteric lymph nodes were isolated from donor mice and processed according to the manufacturer’s protocol. Naïve CD8+ T cells were then either used directly or activated with anti-CD3 (2 μg/ml) and anti-CD28 (1 μg/ml) (both eBioscience) for 16 h and expanded for 6 days in IL-2 (15 ng/ml, in-house produced).
Haas L., Elewaut A., Gerard C.L., Umkehrer C., Leiendecker L., Pedersen M., Krecioch I., Hoffmann D., Novatchkova M., Kuttke M., Neumann T., de Silva I.P., Witthock H., Cuendet M.A., Carotta S., Harrington K.J., Zuber J., Scolyer R.A., Long G.V., Wilmott J.S., Michielin O., Vanharanta S., Wiesner T, & Obenauf A.C. (2021). Acquired resistance to anti-MAPK targeted therapy confers an immune-evasive tumour microenvironment and cross-resistance to immunotherapy in melanoma. Nature cancer, 2(7), 693-708.
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7

CD8+ T cell responses against HPV16 E7

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Autologous CD8+ T cells and LC from HLA-A*0201+ donors were co-cultured in vitro over several weeks to elicit primary CD8+ T cell responses against HPV16 E7 using a previously described protocol [6 (link), 30 (link)]. LC were generated as described above, and untouched CD8+ T cells were purified from PBMC using a negative selection naïve CD8 T cell isolation kit (Miltenyi Biotec, San Diego, CA). LC were left untreated or exposed to HPV16-L1/L2-E7 cVLP, then left untreated or treated with s-Poly-I:C. As a positive control, other LC treated with s-Poly-I:C were exogenously loaded with HLA-A*0201 binding peptides (E711-20, E782-90 and E786-93). CD8+ T cells and LC were co-cultured with irradiated LC at a 20:1 (R:S) ratio for 7 days at 37 °C. Cultures were restimulated with untreated or treated LC at days 7, 14 and 21. After 28 days T cells were harvested and tested for peptide-specific IFN-γ production by ELISPOT.
Da Silva D.M., Woodham A.W., Rijkee L.K., Skeate J.G., Taylor J.R., Koopman M.E., Brand H.E., Wong M.K., McKee G.M., Salazar A.M, & Kast W.M. (2015). Human papillomavirus-exposed Langerhans cells are activated by stabilized Poly-I:C. Papillomavirus Research, 1, 12-21.
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8

Activation and Metabolic Modulation of Naive CD8+ T Cells

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Primary naïve CD8+ T cells were purified from spleens of OT-1 mice using a naïve CD8+ T cell isolation kit (Miltenyi Biotec; cat. 130-096-543). The isolated naïve CD8+ T cells were activated for 72 hours at 37 °C in plates coated with 2 μg/ml anti-CD3 (clone 145-2C11; Bio X Cell; cat. BE0001-1) and 2 μg/ml anti-CD28 (clone 37.51; Bio X Cell; cat. BE0015-1) in the presence of 100 units/ml IL-2 (R&D Systems; cat. 202-IL-050) and 10 ng/ml IL-12 (PeproTech; cat. 210-12-50 mg). CD8+ T cells were cultured either in RPMI-1640 (Thermo Fisher Scientific, cat. 11875-119) supplemented with 10% FBS (MilliporeSigma; cat. F244), 100 units/ml penicillin and 100 mg/ml streptomycin (Thermo Fisher Scientific; cat. 15140-122), 10 mmol/l HEPES (Thermo Fisher Scientific; cat. 15630-130), 1 mmol/l sodium pyruvate (Thermo Fisher Scientific; cat. 11360-070), and 55 mmol/l 2-mercaptoethanol (Thermo Fisher Scientific; cat. 21985–023) or CM. CM was obtained from tumor cells cultured for 24 hours and filtered using 0.2 μm filters. Sodium lactate (Sigma; cat. 867-56-1) was added at the concentration of 10 mM; dimethyl succinate (Sigma; cat. 106-65-0) and dimethyl α-ketoglutarate (Sigma; cat. 349631) were added at the concentration of 500 μM; sodium fumarate (Sigma; cat. F1506), malic acid (Sigma; cat. M7397) and oxaloacetic acid (Sigma; O4126) were added at the concentration of 4 mM.
Elia I., Rowe J., Johnson S., Joshi S., Notarangelo G., Kurmi K., Freeman G.J., Sharpe A.H, & Haigis M.C. (2022). Tumor cells dictate anti-tumor immune responses by altering pyruvate utilization and succinate signaling in CD8+ T cells. Cell metabolism, 34(8), 1137-1150.e6.
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9

Assessing CD8+ T cell proliferation in diabetes

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Donor splenocytes were purified from the spleens of non-diabetic 14w or 24w female mice. Splenocytes (1×107) were intravenously transferred into 6–8w wild type or Tyk2KO.NOD.SCID female recipient mice. Blood glucose levels were measured in tail vein blood once weekly using Glutest Ai (Sanwa). To analyze the proliferation of CD8+ T cells in LNs, naïve 8.3 CD8+ T cells from the LNs of non-diabetic female NY8.3 NOD mice were purified using two rounds of the naïve CD8+ T cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) with LS columns (Miltenyi Biotec). The average purity of the sorted cells was 96%. Purified naïve 8.3 CD8+ T cells were labeled using a CellTrace violet cell proliferation kit (Invitrogen, Waltham, MA, USA) according to the manufacturer’s protocol. Then, 4 ×106 labeled naïve 8.3 CD8+ T cells were transferred into 6–9w or 24w recipient mice. Next, 5 or 10d after the transfer, the PLN and iLN were harvested and the proliferation of CTV labeled cells were analyzed using a FACSVerse flow cytometer (BD Biosciences).
Mine K., Nagafuchi S., Akazawa S., Abiru N., Mori H., Kurisaki H., Shimoda K., Yoshikai Y., Takahashi H, & Anzai K. (2024). TYK2 signaling promotes the development of autoreactive CD8+ cytotoxic T lymphocytes and type 1 diabetes. Nature Communications, 15, 1337.
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10

Naive CD8+ T Cell Stimulation

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Naïve CD8+ T cells were negatively isolated using the Naïve CD8+ T Cell Isolation Kit (Miltenyi Biotec, catalog #130-096-543) according to the manufacturer’s instructions. Cells were labeled using the CellTrace Violet Cell Proliferation Kit (CTV; Termo Fisher Scientifc) and 5x104 cells were stimulated with coated aCD3 (0.2 to 5 µg/mL, BioXCell clone 145-2C11, catalog #BE0001-1) and aCD28 (0.08 to 2 µg/mL, BioXCell clone 37.51, catalog #BE0015-1) antibodies in complete RPMI with murine IL-7 (2.5 ng/mL, Peprotech, catalog #217-17) +/- murine IL-2 (5 ng/mL, Miltenyi Biotec, catalog #130-120-332). After 4 days of culture, supernatants were harvested and stored at -80°C until further use and proliferation and cytokine expression were assessed by FACS.
Voisin A., Plaschka M., Perrin-Niquet M., Twardowski J., Boutemine I., Eluard B., Lalle G., Stéphan P., Bouherrou K., Tonon L., Pommier R., Ferrari A., Klein U., Wencker M., Baud V., Cassier P.A, & Grinberg-Bleyer Y. (2024). The NF-κB RelA transcription factor is not required for CD8+ T-cell function in acute viral infection and cancer. Frontiers in Immunology, 15, 1379777.
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