CD8+ T lymphocytes were purified from splenocytes using a CD8+ isolation kit (Miltenyi Biotec). Purified CD8+ T lymphocytes were cultured in the presence of plate-bound anti-CD3 (10 µg/ml) with or without recombinant murine VEGF-A (50 ng/ml; Miltenyi Biotec). After 48 h of culture, cells were harvested and analyzed by cytometry or used to extract mRNA. In some experiments, anti–VEGF-R1 (20 µg/ml; R&D Systems) or anti–VEGF-R2 (10 µg/ml; clone 91202; R&D Systems) antibodies or isotype control were added to the culture medium. In some experiments, 11R-VIVIT (Merck Millipore) was added 1 h at 5 µM before the addition of VEGF-A and during the stimulation with VEGF-A.
Cd8 isolation kit
Manufactured by Miltenyi Biotec
Sourced in United States
The CD8 isolation kit is a laboratory tool used to isolate and purify CD8+ T cells from a sample. It utilizes magnetic bead-based technology to selectively separate CD8+ cells from the rest of the cell population in a sample.
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Lab products found in correlation
Violetdye, by Thermo Fisher Scientific (2 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (2 mentions)
Anti biotin bead, by Miltenyi Biotec (2 mentions)
Biotinylated anti mouse cd44, by BioLegend (2 mentions)
Ionomycin, by Merck Group (2 mentions)
Anti vegfr1, by R&D Systems (1 mentions)
Anti vegfr2, by R&D Systems (1 mentions)
11r vivit, by Merck Group (1 mentions)
Vegf a, by Merck Group (1 mentions)
Anti cd3, by Miltenyi Biotec (1 mentions)
Mouse mdscs isolation kit, by Miltenyi Biotec (1 mentions)
Lsrfortessa flow cytometer, by BD (1 mentions)
Live dead fixable aqua dye, by Thermo Fisher Scientific (1 mentions)
Retronectin, by Takara Bio (1 mentions)
Recombinant human interleukin 7, by Thermo Fisher Scientific (1 mentions)
Concanavalin a (cona), by Merck Group (1 mentions)
Anti cd3 cd28, by Thermo Fisher Scientific (1 mentions)
Anti cd3, by Thermo Fisher Scientific (1 mentions)
Cycloheximide (chx), by Merck Group (1 mentions)
Actinomycin d, by Merck Group (1 mentions)
19 protocols using cd8 isolation kit
1
VEGF-A Modulation of CD8+ T Cell Activity
2
Adoptive Transfer of Naïve OT-1 T Cells
3
Isolation and Characterization of Mouse Myeloid-Derived Suppressor Cells
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For mouse BMDCs, femurs were obtained from 4–6-wk-old C57BL/6 mice, and bone marrow was flushed aseptically with RPMI medium using a syringe fitted with a 27-gauge needle. Mouse CD8+ T cells were isolated from mouse spleen using a CD8+ isolation kit (Miltenyi Biotec; 130-104-075). MDSCs were isolated from spleens of tumor-bearing mice using a mouse MDSCs isolation kit (Miltenyi Biotec; 130-094-538). Cell purity was checked by flow cytometric analysis using anti-CD11b and Gr-1 antibodies (>95%), and cell viability was checked by Trypan blue dye exclusion. The resulting cells were cultured using RPMI medium supplemented with 10% FBS at 37°C, 5% CO2, and used for further tumor-induced MDSC conversion, MDSC expansion, and MDSC function assays.
For the mouse tumor-induced MDSC conversion assay, BMDCs were derived from healthy 4–6-wk-old C57BL/6 mice and cocultured with PANC02-vector/PANC02-EHF. After 6-d coculture, cells were collected and stained for CD45, CD11b, and Gr-1. For mouse MDSC expansion assays, isolated MDSCs were stained with CFSE. Then, labeled MDSCs were cocultured with PANC02-vector/PANC02-EHF at 5:1 for 72 h. Proliferation was assessed by measuring CFSE dilution by flow cytometry.
For the mouse tumor-induced MDSC conversion assay, BMDCs were derived from healthy 4–6-wk-old C57BL/6 mice and cocultured with PANC02-vector/PANC02-EHF. After 6-d coculture, cells were collected and stained for CD45, CD11b, and Gr-1. For mouse MDSC expansion assays, isolated MDSCs were stained with CFSE. Then, labeled MDSCs were cocultured with PANC02-vector/PANC02-EHF at 5:1 for 72 h. Proliferation was assessed by measuring CFSE dilution by flow cytometry.
4
Isolation and Activation of Murine CD8+ T Cells
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Single-cell suspensions were obtained from inguinal and mesenteric lymph nodes from healthy C57BL/6 naïve mice (6–12 weeks old, 18–20 g) kept at the Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa. CD8+ cells were isolated using the CD8+ isolation kit from Miltenyi Biotec (USA). Cells were CTV-labeled and stimulated in vitro with 5 µg/mL of recombinant Fc-fusion proteins containing hIL-2-derived beta super-binders from phage libraries. Cells treated with similarly formatted H9 superkine- and hIL-2-containing fusion proteins were included as references. Non-stimulated cells were analyzed as negative control (basal proliferation). For flow cytometry analysis, dead cells were excluded with live/dead fixable aqua dye (Thermo Fisher Scientific, USA), and fluorescence dilution was determined on a Beckton Dickinson LSR Fortessa flow cytometer. Acquired data were analyzed with FlowJo software (Tree Star).
5
Expansion of Antigen-Specific CD8 T Cells
6
Induction of Vitiligo in Mice
7
Adoptive Transfer of Naïve OT-1 T Cells
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The CD8+ T cells were isolated from
Rag−/− OT-1 TCR transgenic mice using a CD8
isolation kit (Miltenyi Biotec, Auburn, CA). The CD44+ cells
were depleted using biotinylated anti-mouse CD44 (1:2000 dilution, BioLegend,
San Diego, CA) and anti-biotin beads (Miltenyi Biotec). The naïve
CD8+ OT-1 T cells were labeled with either CFSE or violet
dye (both from Invitrogen, Grand Island, NY) at 5 μM. Ten microliters of
CFSE-labeled cells (1×108 ml−1) were
directly injected into the vaginal tissue (Ivag) using a Hamilton syringe with a
32G needle. One hundred microliters of violet dye-labeled cells
(1×107 ml−1) were injected into the
tail vein (IV). The mice were under isoflurane general anesthesia during the
performance of dual transfer.
Rag−/− OT-1 TCR transgenic mice using a CD8
isolation kit (Miltenyi Biotec, Auburn, CA). The CD44+ cells
were depleted using biotinylated anti-mouse CD44 (1:2000 dilution, BioLegend,
San Diego, CA) and anti-biotin beads (Miltenyi Biotec). The naïve
CD8+ OT-1 T cells were labeled with either CFSE or violet
dye (both from Invitrogen, Grand Island, NY) at 5 μM. Ten microliters of
CFSE-labeled cells (1×108 ml−1) were
directly injected into the vaginal tissue (Ivag) using a Hamilton syringe with a
32G needle. One hundred microliters of violet dye-labeled cells
(1×107 ml−1) were injected into the
tail vein (IV). The mice were under isoflurane general anesthesia during the
performance of dual transfer.
8
Murine T Cell Activation and Transduction
9
ChIP Assay of Activated CD8+ T Cells
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A total of 107 CD8+T cells, isolated from human PBMC using CD8 isolation kit (Miltenyi Biotec), was activated with plate-bound anti-CD3 (1 ug/ml) and anti-CD28 (1 ug/ml) in 96-well plates for 72 h. Chromatin immunoprecipitation (ChIP) assays were performed according to the protocol of the SimpleChIP® Plus Sonication Chromatin IP Kit (56,383). NR4A1 antibody used for ChIP was purchased from Novus Biologicals. DNA isolated was tested by real-time quantitative PCR. The enrichment of samples in chromatin was based on the calculation formula, △CT = CTChIP DNA – CTInput DNA. Specific information on antibodies was listed in Table. S1.
10
Evaluating Anti-tumor T-cell Responses
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Tumor cells were killed and dead cells were harvested from suspension and counted, centrifuged for 10 min at 1000×g, and 3×105 or 1×105 cells were added to 105 differentiated BM-DC per well in a 96-well round bottom plate. After 18 hours BM-DC were centrifuged for 3 min at 500×g, rinsed with PBS, centrifuged again and resuspended in complete RPMI medium. 2C or OT-I T cells were harvested from spleens of transgenic Rag2-/- mice and isolated by magnetic separation using CD8+ isolation kit (Miltenyi) before counting and staining with proliferation dye CellTrace Violet (Invitrogen). OT-I T cells (5×105) were added to each well and cultured for 72 hours before staining and analysis by flow cytometry. BM-DC without dead B16F10 or media supplemented with 1 µg/mL ionomycin (Sigma Aldrich) and 100 ng/mL phorbol 12-myristate 13-acetate (Sigma Aldrich) served as negative and positive controls for T-cell activation, respectively. For assays using B2M−/− B16F10 cell lines, total cells (both adherent and suspended) were isolated by trypsinization, centrifuged for 10 min at 1000×g, and added to BM-DC. For assays using doxocycline, vincristine, and palbociclib, B16F10 cells were washed twice with PBS, each time pelleting by centrifugation for 10 min at 1000×g prior to addition to BM-DC cultures.
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