Spleen and lymph nodes of JEDI mice were dissected, and single cell suspensions of leukocytes were obtained by mechanical disruption and filtering through a 70-mm cell strainer. Red blood cells were lysed using ACK buffer (Lonza), and CD8+ T cells were negatively selected using MagniSort Mouse CD8+ T Cell Enrichment Kit (ThermoFisher Scientific) according to the manufacturer’s protocol. For in vitro experiments described in the text using JEDI, cells were activated for 2 days with 5 μg/mL plate-bound purified hamster anti-mouse CD3e (clone 145–2C11, BD Biosciences), 1 μg/mL purified hamster anti-mouse CD28 (clone 37.51, BD Biosciences), and 20 ng/mL recombinant mouse IL-2 (Gemini Bio-Products) in RPMI with 10% FBS, 100 U/ml penicillin/streptomycin/ampicillin, and 50 μM 2-mercaptoethanol. For in vitro experiments described in the text using GFP-specific T cells, cells were first FACS purified using H-2Kd-HYLSTQSAL tetramer reagent produced by the NIH Tetramer Core Facility prior to activation as described above. For all in vivo experiments, freshly isolated tetramer-sorted GFP-specific T cells were used without activation.
Magnisort mouse cd8 t cell enrichment kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The MagniSort Mouse CD8+ T Cell Enrichment Kit is a magnetic-based separation system designed to isolate CD8+ T cells from mouse splenocytes or lymph node cells. The kit utilizes magnetic beads coated with antibodies specific to the CD8 molecule, allowing for the efficient separation of CD8+ T cells from the cell suspension.
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Lab products found in correlation
2 mercaptoethanol, by Thermo Fisher Scientific (2 mentions)
Celltrace violet, by Thermo Fisher Scientific (2 mentions)
Hamster anti mouse cd28 clone 37.51, by BD (1 mentions)
Ack buffer, by Lonza (1 mentions)
Complete rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Siinfekl peptide, by GenScript (1 mentions)
Cytotox 96 non radioactive cytotoxicity assay kit, by Promega (1 mentions)
Recombinant ifn β, by R&D Systems (1 mentions)
Lsrfortessa x 20, by BD (1 mentions)
Annexin 5 fitc apoptosis detection kit, by Vazyme (1 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
Mem non essential amino acid, by Thermo Fisher Scientific (1 mentions)
Flt3l, by Thermo Fisher Scientific (1 mentions)
Interleukin 4 (il 4), by Thermo Fisher Scientific (1 mentions)
Gm csf, by Thermo Fisher Scientific (1 mentions)
Hepes, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Soluble anti cd28 37.51, by BD (1 mentions)
Cd80 fc, by BioLegend (1 mentions)
10 protocols using magnisort mouse cd8 t cell enrichment kit
1
Isolation and Activation of CD8+ T Cells
2
Adoptive Transfer of CD8 Cells
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For the adoptive transfer of CD8 cells into C57BL/6 hosts with long-surviving BALB/c allografts, WT or M290-MC-MMAF-treated mice (6-week-old males) that were to be used as lymphocyte donors were i.p. injected with 10 × 106 BALB/c splenocytes. On day 6, the spleens and abdominal lymph nodes were harvested and centrifuged on Lympholyte-M to remove RBCs. The resulting cell suspension was enriched for CD8+ T cells by immunomagnetic bead sorting (MagniSort™ Mouse CD8 T cell Enrichment Kit, Thermo Fisher Scientific, Carlsbad, CA, USA). FACS analyses using anti-CD8, anti-CD3, and anti-CD103 antibodies were performed on each preparation to assess the degree of enrichment for CD8+ T cells and the expression of CD103. A total of 50 × 106 CD8+ T lymphocytes were injected into C57BL/6 mice bearing long-surviving BALB/c islets via the tail vein, and blood glucose was monitored daily to assess graft survival.
3
CD8+ T Cell Proliferation and Cytotoxicity Assay
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Single cell suspensions were prepared from spleen and lymph nodes of JEDI mice, and CD8+ T cells were negatively selected using MagniSort Mouse CD8+ T Cell Enrichment Kit (ThermoFisher Scientific) according to the manufacturer’s protocol. To analyze proliferation, some experiments were performed with splenocytes stained with CellTrace Violet (ThermoFisher Scientific) according to the manufacturer’s protocol. T cell killing assays were performed by co-culturing 10,000 GFP+ A20 cells, 10,000 mCherry+ A20 cells and naïve bulk JEDI splenocytes (splenocyte:tumor ratios of 5:1 to 20:1) or isolated JEDI CD8+ T cells (T cell:tumor ratios of 1:1 to 5:1) in 96-well U-bottom plates. Cells were harvested for analysis by flow cytometry after 2–5 days of culture.
4
Enrichment and Migration of CD8+ T Cells
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MagniSort™ Mouse CD8 + T Cell Enrichment Kit (8804-6822-74, ThermoFisher, USA) was used to isolate CD8 + T cells from breast cancer mouse tumor tissue. The tumor tissue from the orthotopic breast cancer mouse model was treated to obtain tumor extracellular matrix (ECM), and then ECM and CD8 + T cells (5 × 105 cells) were placed in the upper chamber. CCL21 chemokine (7338–50, Wuhan Aimijie Technology Co., Ltd., Wuhan) was added to the lower chamber tumor-conditioned medium, and the cells were moved for 2 h at 37 °C. Flow cytometry was used to detect the number of CD8 + T cells that migrated to the lower chamber, and the number of CD8 + T migration cells without ECM and CCL12 was set as “1”.
5
Cytotoxicity Assay for OT-I T Cell-Mediated Killing
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OT-I T cells were isolated from the spleen and lymph nodes of 8-week-old OT-I mice (purchased from The Jackson Laboratory) using MagniSort Mouse CD8 T-Cell Enrichment Kit (Thermo Fisher Scientific) according to the manufacturer’s guidance. OT-I T cells were maintained in complete RPMI 1640 (Gibco) supplemented with 2-mercaptoethanol (Gibco) or treated with SIINFEKL peptide (GenScript) for OT-I T cell activation. For cytotoxic T lymphocyte assay, the B16-OVA or ID8-OVA tumor cells were transfected with siNC or siRAD21 for 48 hours and then treated with or without recombinant IFN-β (R&D Systems) for another 24 hours. The pretreated cells were then cocultured with activated OT-I T cells at a ratio of 1:1 for 48 hours. Apoptotic cells were quantified using the Annexin V–FITC Apoptosis Detection Kit (Vazyme) according to the manufacturer’s protocol and analyzed with a BD LSRFortessa X-20 (BD Biosciences). The LDH release was determined using CytoTox96 Non-Radioactive Cytotoxicity Assay Kit (Promega) following the manufacturer’s instructions, and percentage cytotoxicity was calculated as per a previous study (59 (link)).
6
Isolation and Culture of BMDCs and OT-I T Cells
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BMDCs were generated by isolating bone marrow cells from 6-to 8-week-old female mice and cultured with GMCSF and IL4 (Peprotech, 315-03, 214-14, 20 ng/mL) or with FLT3L (Peprotech, 250-31L, 100 ng/mL). The culture media was refreshed every 2 days and BMDCs were used 7 days after culture. CD8 þ OT-I T cells were isolated from the spleen and lymph nodes of 8-week-old OT-I mice using MagniSort Mouse CD8 T-Cell Enrichment Kit (Thermo Fisher Scientific, 8804-4622-74). BMDCs and OT-I T cells were both cultured in RPMI1640 (Gibco, 11875-176) supplemented with 10% FBS, 1% penicillin-streptomycin and 55 mmol/L 2-mercaptoethanol (Gibco, 21985023). BMDCs were also supplemented with MEM Nonessential Amino Acid (Gibco, 11140050), HEPES (Gibco, 15630130), and sodium pyruvate (Gibco, 11360070) in a humidified incubator at 37 C and 5% CO 2 .
7
Stimulation of CD8 T-cells with PD-L1 and CD80
8
CD8 T Cell Isolation and Activation
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CD8 T cells were isolated from LNs or spleens using the MagniSort Mouse CD8 T Cell Enrichment Kit (Invitrogen) according to the manufacturer’s instructions. For activation assays, isolated CD8 T cells, total LN cells, or total spleen cells were cultured in anti-CD3–coated (1 µg/ml; Sigma-Aldrich) cell culture plates in the presence of IL-2 (5 ng/ml) and anti-CD28 (3 µg/ml; Sigma-Aldrich) for 48 h, unless otherwise stated.
9
Coculture of Mouse CD8+ T Cells and LLC Cells
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A mouse Lewis lung carcinoma (LLC) cell line and mouse peripheral blood mononuclear cells (PBMCs) were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). LLC cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, USA) at 37 °C under 5% CO2.
PBMCs were cultured in RPMI-1640 medium (Gibco, USA) supplemented with 10% FBS (Gibco, USA) at 37 °C under 5% CO2. A MagniSort™ Mouse CD8+T-cell Enrichment Kit (Invitrogen, USA) was used for negative selection of mouse CD8+ T cells. Then, CD8+ T cells were activated with Dynabeads™ Mouse T-Activator CD3/CD28 (Gibco, USA). CD8+ T cells and LLC cells were cocultured for 48 h. In brief, activated CD8+ T cells were resuspended in serum-free RPMI 1640 medium in the upper chamber of a transwell plate (5 mm), and LLC cells were cultured in the lower chamber of a transwell chamber.
PBMCs were cultured in RPMI-1640 medium (Gibco, USA) supplemented with 10% FBS (Gibco, USA) at 37 °C under 5% CO2. A MagniSort™ Mouse CD8+T-cell Enrichment Kit (Invitrogen, USA) was used for negative selection of mouse CD8+ T cells. Then, CD8+ T cells were activated with Dynabeads™ Mouse T-Activator CD3/CD28 (Gibco, USA). CD8+ T cells and LLC cells were cocultured for 48 h. In brief, activated CD8+ T cells were resuspended in serum-free RPMI 1640 medium in the upper chamber of a transwell plate (5 mm), and LLC cells were cultured in the lower chamber of a transwell chamber.
10
Activation of Naive CD8+ T Cells
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WT CD8 þ T cells were isolated from spleens and lymph nodes using the MagniSort mouse CD8 T-cell enrichment kit (Invitrogen). Naive CD8 þ CD44 lo CD62L hi cells were sorted from WT to >90% purity with a BD FACS Aria II (BD Biosciences). Naive CD8 þ T cells were resuspended at 1 Â 10 6 cells/mL in complete medium in the presence or absence of anti-CD3 (10 mg/mL, eBioscience) and anti-CD28 (2 mg/mL, BD Biosciences) and incubated on ice for 30 minutes. Rabbit anti-hamster IgG (secondary cross-linking Ab, 1 mg/ml, Sigma-Aldrich) was added, and cells were transferred to 37 C for the indicated times. Stimulations were terminated by washing twice with cold PBS.
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