Easysep mouse cd8α positive selection kit, by STEMCELL - Product details

1

Assessing CD11c+ and CD8+ T cell function

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For bone-marrow CD11c⁺ cells functional assay, 2×10⁴ purified CD11c⁺ cells with were incubated with isolated CD8⁺ T cells from naive 2C mice with EasySep™ Mouse CD8α Positive Selection Kit (STEMCELL) for three days at the ratio of 1:10. For tumor-specific CD8⁺ T cells functional assay, eight days after radiation, tumor DLNs were removed and CD8⁺ T cells were purified. MC38 tumor cells were exposed to 20ng/ml murine IFN-γ for 24 hr prior to plating with purified CD8⁺ T. 2×10⁵ CD8⁺ T cells were incubated with MC38 at the ratio of 10:1 for 48 hours. ELISPOT assays were performed to detect the cytokine spots of IFN-γ according to product protocol (Millipore).

Deng L., Liang H., Xu M., Yang X., Burnette B., Arina A., Li X.D., Mauceri H., Beckett M., Darga T., Huang X., Gajewski T.F., Chen Z.J., Fu Y.X, & Weichselbaum R.R. (2014). STING-dependent Cytosolic DNA Sensing Promotes Radiation-induced Type I interferon-dependent Antitumor Immunity in Immunogenic Tumors. Immunity, 41(5), 843-852.

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2

Neoantigen-specific CD8+ TIL Activation

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Either 1x10⁵ splenocytes or 1x10⁴ CD8⁺ TILs, isolated from tumours using the EasySep Mouse CD8α Positive Selection Kit (Stemcell Technologies), were harvested from tumour-bearing mice and pulsed with 1μM peptide corresponding to clonal neoantigens predicted from KPAR1.3 WES (Supplementary Table 1) or eMLV env peptide (KSPWFTTL). TILs were co-incubated with 1x10⁵ splenocytes from naïve mice as a source of dendritic cells. Cells were stimulated for 24h in anti-mouse IFNγ-coated ELISpot plates (BD Bioscience). Plates were developed according to manufacturer’s instructions and quantified using a CTL S6 machine.

Boumelha J., de Carné Trécesson S., Law E.K., Romero-Clavijo P., Coelho M.A., Ng K., Mugarza E., Moore C., Rana S., Caswell D.R., Murillo M., Hancock D.C., Argyris P.P., Brown W.L., Durfee C., Larson L.K., Vogel R.I., Suárez-Bonnet A., Priestnall S.L., East P., Ross S.J., Kassiotis G., Molina-Arcas M., Swanton C., Harris R, & Downward J. (2022). An immunogenic model of KRAS-mutant lung cancer enables evaluation of targeted therapy and immunotherapy combinations. Cancer research, 82(19), 3435-3448.

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3

OT-I CD8+ T cell proliferation assay

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Mouse splenic CD8⁺ T cells from OT-I mice were isolated using EasySep Mouse CD8α Positive Selection Kit (STEMCELL Technologies). Isolated OT-I T cells were labeled with CellTrace Violet (CTV) Cell proliferation Kit (Thermo Fisher) manufacturer instruction. The CTV-labeled CD8⁺ T cells (0.5 x 10⁵ per well) were cultured in plate-bound 1 μg/ml anti-CD3 (clone 145-2C11; Thermo Fisher) and 5 μg/ml anti-CD28 (Clone 37.51; Thermo Fisher) with complete RPMI media containing 55 μM β-mercaptoethanol and 10 ng/ml IL-2. IL-4 or LPS+IFNγ stimulated macrophages were then added into T cell cultures with ratio 1:10 (macrophages:T cells) for 72 h. Cells were then harvested and CTV positive signal in the CD8⁺ gate was measured by flow cytometry.

Raines L.N., Zhao H., Wang Y., Chen H.Y., Gallart-Ayala H., Hsueh P.C., Cao W., Koh Y., Alamonte-Loya A., Liu P.S., Ivanisevic J., Lio C.W., Ho P.C, & Huang S.C. (2022). PERK is a critical metabolic hub for immunosuppressive function in macrophages. Nature immunology, 23(3), 431-445.

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4

Isolation and Sorting of Intestinal T-IEL

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T-IEL were isolated for sorting from mice and as described in James et al., 2020 (link). Briefly, small intestines were extracted and flushed. Small intestines were longitudinally opened, then transversely cut into ~5 mm pieces and put into warm media containing 1 mM DTT. Small intestine pieces were shaken for 40 min, centrifuged, vortexed, and passed through a 100 μm sieve. The flow-through was centrifuged on a 36%/67% Percoll density gradient at 700 g for 30 min. The T-ΙEL were isolated from the interface between 36% and 67% Percoll. In some experiments, isolated T-IEL were further enriched using an EasySep Mouse CD8α-positive selection kit (STEMCELL Technologies) as per the manufacturer’s instructions. Isolation and sorting details for the LN and effector populations used for proteomics can be found at http://www.immpres.co.uk/ under the ‘Protocols & publications’ tab.

Brenes A.J., Vandereyken M., James O.J., Watt H., Hukelmann J., Spinelli L., Dikovskaya D., Lamond A.I, & Swamy M. (2021). Tissue environment, not ontogeny, defines murine intestinal intraepithelial T lymphocytes. eLife, 10, e70055.

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5

Pmel-1 CD8+ T Cell Activation Assay

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CD8⁺ cells were isolated from Pmel-1 splenocytes using EasySep Mouse CD8α Positive Selection Kit (STEMCELL Technologies). The isolated cells were labeled with CellTrace carboxyfluorescein succinimidyl ester (CFSE) (Thermo Fisher Scientific), and seeded onto 24-well plate (2×10⁶ cells/well) in media containing interleukin (IL)-2 (60 IU/mL) (Prometheus Laboratories) with H-2D^b-restricted epitope of the influenza nucleoprotein (NP) peptide, NP_366–374 (ASNENMETM; GenScript) (1 µM) or human (h)gp100_25–33 peptide (KVPRNQDWL: GenScript) (1 µM). Then, iPSC-DCs (1×10⁶) stimulated with mouse IL-4 (mIL-4) (Peprotech) (10 ng/mL), mouse tumor necrosis factor alpha (mTNFα) (Peprotech) (5 ng/mL), and lipopolysaccharides (LPS) (Sigma-Aldrich) (0.5 µg/mL) for 2 days were added to each well and co-cultured for 2 days. In vitro-activated Pmel-1 CD8⁺ T cells were evaluated for proliferation, surface markers, and cytokine production against NP_366–374 and hgp100_25–33 peptides.

Oba T., Makino K., Kajihara R., Yokoi T., Araki R., Abe M., Minderman H., Chang A.E., Odunsi K, & Ito F. (2021). In situ delivery of iPSC-derived dendritic cells with local radiotherapy generates systemic antitumor immunity and potentiates PD-L1 blockade in preclinical poorly immunogenic tumor models. Journal for Immunotherapy of Cancer, 9(5), e002432.

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6

Assessing CD8+ T Cell Activation

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Eight days after treatment, tumor DLNs were removed and CD8⁺ T cells were purified with EasySep Mouse CD8α Positive Selection Kit (StemCell). 2 × 10⁵ CD8⁺ T cells were incubated with 1 × 10⁵ irradiated (12 Gy) splenocytes from naive mice in the presence or absence of 1 μg/ml SIY peptide for 48 hours. ELISPOT assays were performed to detect the cytokine spots of IFN-γ according to product protocol (Millipore).

Zheng W., Skowron K.B., Namm J.P., Burnette B., Fernandez C., Arina A., Liang H., Spiotto M.T., Posner M.C., Fu Y.X, & Weichselbaum R.R. (2016). Combination of radiotherapy and vaccination overcomes checkpoint blockade resistance. Oncotarget, 7(28), 43039-43051.

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7

CD11c+ and CD8+ T Cell Functional Assays

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For bone-marrow CD11c⁺ cells functional assay, 2×10⁴ purified CD11c⁺ or F4/80⁺ cells with were incubated with isolated CD8⁺ T cells from naive OT-I mice with EasySep™ Mouse CD8α Positive Selection Kit (STEMCELL) for three days at the ratio of 1:10. For tumor-specific CD8⁺ T cells functional assay in MC38-OTI model, 5 days after anti-CD47 mAb treatment, tumor DLNs were removed and CD8⁺ T cells were purified. 2×10⁵ CD8⁺ T cells were incubated with BMDC at the ratio of 10:1 for 48 hours with/out 5µg/ml OTI peptide (SIINFEKL). For tumor-specific CD8⁺ T cells functional assay in MC38 and A20 models, 5 days after anti-CD47 Ab treatment, tumor DLNs were removed. DLN cells were re-stimulated with A20 or MC38. 96-well HTS-IP plate (Millipore) was pre-coated with anti-IFN-γ antibody (ebiosceince) with a 1:250 dilution overnight at 4 °C. After co-culture, cells were removed, 2 µg/ml biotinylated anti-IFN-γ antibody (ebiosceince) with a 1:250 dilution was added, and the plate was incubated for 2h at room temperature or overnight at 4 °C. Avidin-horseradish peroxidase (BD Pharmingen) with a 1:1000 dilution was then added and the plate was incubated for 1h at room temperature. The cytokine spots of IFN-γ were developed according to product protocol (Millipore).

Liu X., Pu Y., Cron K., Deng L., Kline J., Frazier W.A., Xu H., Peng H., Fu Y.X, & Xu M.M. (2015). CD47 Blockade Triggers T cell-mediated Destruction of Immunogenic Tumors. Nature medicine, 21(10), 1209-1215.

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8

CD11c+ and CD8+ T Cell Functional Assays

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For bone-marrow CD11c⁺ cells functional assay, 2×10⁴ purified CD11c⁺ or F4/80⁺ cells with were incubated with isolated CD8⁺ T cells from naive OT-I mice with EasySep™ Mouse CD8α Positive Selection Kit (STEMCELL) for three days at the ratio of 1:10. For tumor-specific CD8⁺ T cells functional assay in MC38-OTI model, 5 days after anti-CD47 mAb treatment, tumor DLNs were removed and CD8⁺ T cells were purified. 2×10⁵ CD8⁺ T cells were incubated with BMDC at the ratio of 10:1 for 48 hours with/out 5µg/ml OTI peptide (SIINFEKL). For tumor-specific CD8⁺ T cells functional assay in MC38 and A20 models, 5 days after anti-CD47 Ab treatment, tumor DLNs were removed. DLN cells were re-stimulated with A20 or MC38. 96-well HTS-IP plate (Millipore) was pre-coated with anti-IFN-γ antibody (ebiosceince) with a 1:250 dilution overnight at 4 °C. After co-culture, cells were removed, 2 µg/ml biotinylated anti-IFN-γ antibody (ebiosceince) with a 1:250 dilution was added, and the plate was incubated for 2h at room temperature or overnight at 4 °C. Avidin-horseradish peroxidase (BD Pharmingen) with a 1:1000 dilution was then added and the plate was incubated for 1h at room temperature. The cytokine spots of IFN-γ were developed according to product protocol (Millipore).

Liu X., Pu Y., Cron K., Deng L., Kline J., Frazier W.A., Xu H., Peng H., Fu Y.X, & Xu M.M. (2015). CD47 Blockade Triggers T cell-mediated Destruction of Immunogenic Tumors. Nature medicine, 21(10), 1209-1215.

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Easysep mouse cd8α positive selection kit

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Close spelling variants of this product

8 protocols using easysep mouse cd8α positive selection kit

Assessing CD11c+ and CD8+ T cell function

Neoantigen-specific CD8+ TIL Activation

OT-I CD8+ T cell proliferation assay

Isolation and Sorting of Intestinal T-IEL

Pmel-1 CD8+ T Cell Activation Assay

Assessing CD8+ T Cell Activation

CD11c+ and CD8+ T Cell Functional Assays

CD11c+ and CD8+ T Cell Functional Assays

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