CD8+ T cells were isolated from peripheral blood mononuclear cell preparations from leukapheresis blood cones (NHS-BT) using the EasySep Human CD8+ T Cell Isolation Kit (Stemcell, Cambridge, MA). CD8+ T cells were then cryopreserved with CryoStor CS5 (Stemcell) prior to activation. Cells were recovered and activated with two rounds of anti-CD3/CD28 stimulation as described in Nelson et al., to generate effector T cells with high cytotoxic capacity. Briefly, 25 µL anti-CD3/CD28 DynaBeads (ThermoFisher, Waltham, MA) per 1 × 10 6 cells were added in the presence of human IL2 at 20 ng/mL (Stemcell). The T cells were maintained at a density of 1 × 10 6 cells/mL. After 10 d, the T cells were reactivated with 25 µL anti-CD3/CD28 DynaBeads (ThermoFisher) per 1 × 10 6 cells in the presence of fresh human IL2 (20 ng/mL) prior to cryopreservation at day 13 with CryoStorCS10 (Stemcell). T cells were recovered from cyropreservation into prewarmed T cell medium and allowed to recover for 2 d before being used in assays.
Human il 2
Manufactured by STEMCELL
Human IL-2 is a recombinant protein that corresponds to the interleukin-2 cytokine produced by T cells. It is a key regulator of immune responses.
Automatically generated - may contain errors
Lab products found in correlation
Recombinant murine il 2, by BioLegend (2 mentions)
Recombinant murine il 4, by BioLegend (2 mentions)
Easysep human cd4 cd127lowcd25 regulatory t cell isolation kit, by STEMCELL (2 mentions)
Pma ionomycin, by BioLegend (2 mentions)
Immunocult cd3 cd28 t cell activator, by STEMCELL (2 mentions)
Easysep mouse cd4 cd25 regulatory t cell isolation kit, by STEMCELL (2 mentions)
Immunocult xf t cell expansion medium, by STEMCELL (2 mentions)
Brefeldin a, by BioLegend (2 mentions)
Jurkat t cells, by American Type Culture Collection (1 mentions)
Immunocult human cd3 cd28 t cell activator, by STEMCELL (1 mentions)
Human cd8 t cell isolation kit, by Miltenyi Biotec (1 mentions)
Mouse cd8 t cell isolation kit, by Miltenyi Biotec (1 mentions)
T cell expansion medium, by STEMCELL (1 mentions)
Mouse il 2, by Miltenyi Biotec (1 mentions)
C57bl 6 mice, by Miltenyi Biotec (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Wisent (1 mentions)
Enh204 fp, by Kerafast (1 mentions)
Anti cd3 cd28 dynabeads, by Thermo Fisher Scientific (1 mentions)
5 protocols using human il 2
1
Generating Cytotoxic CD8+ T Cells
2
Crbn Knockout Regulatory T Cell Culture
3
Isolation and Culture of Immune Cell Lines
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U937 monocytes and Jurkat T cells were obtained from ATCC (Manassas, VA, USA). The U937 and Jurkat cells were cultured in RPMI 1640 medium with 10% fetal bovine serum (FBS). HEK293T cells were kindly provided by Dr. H. John Sharifi (ACPHS) and were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% FBS. Primary lymphocytes were purchased from the Elutriation Core Facility at University of Nebraska Medical Center. The lymphocytes were cultured with RPMI 1640 supplemented with 5 mM glucose and 10% FBS and were differentiated into T cells with ImmunoCult Human CD3/CD28 T Cell Activator and human IL-2 as per manufacturer’s instructions (Stemcell Technologies Inc.). All cells were cultured at 37 °C and 5% CO2.
4
Crbn Knockout Regulatory T Cell Culture
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FLP293T derived stable cell lines were cultured in DMEM supplemented with 10% dialyzed fetal bovine serum (FBS). Wildtype and Crbn−/− Jurkat cells were cultured in RPMI supplemented with 10% FBS and 1% penicillin/streptomycin in a 37°C incubator with 5% CO2. CD4+ CD25+ regulatory T cells were purified from spleens from wildtype or CrbnI391V/I391V mice using the EasySep Mouse CD4+ CD25+ Regulatory T cell Isolation Kit (StemCell Technologies). T cells were cultured in RPMI supplemented with 10% FBS, 1% penicillin/streptomycin, 1% non-essential amino acids, 1% sodium pyruvate, 2 mM GlutaMax, 10 mM HEPES, and 50 μM 2-mercaptoethanol, as well as 5 ng/ml recombinant murine IL-2 (Biolegend) and 20 ng/ml recombinant murine IL-4 (Biolegend). Human regulatory T cells were isolated from peripheral blood mononuclear cells using the EasySep Human CD4+CD127lowCD25+ Regulatory T Cell Isolation Kit (StemCell Technologies) and expanded for 12–14 days in the presence of 500 U/ml human IL-2, Immunocult CD3/CD28 T cell activator (StemCell Technologies), and compound in ImmunoCult-XF T Cell Expansion Medium (StemCell Technologies). Cells were stimulated with 1X PMA/ionomycin (Biolegend) for 1h followed by the addition of brefeldin A (Biolegend) for another 3h.
5
Culturing Human and Mouse Cancer Cells
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Human NSCLC cell lines H1975 (CRL-5908, ATCC), H460 (HTB-177, ATCC), and PC9 (90071810, Sigma) were cultured in RPMI medium (350-007-CL, Wisent) while mouse colon cancer cell line MC38 (ENH204-FP, Kerafast) were cultured in DMEM (D5796, Sigma). All culture media were supplemented with 10% FBS (Wisent), 1% penicillin-streptomycin (Sigma), and incubated at 37 °C with 5% CO2. For the stimulation of cells with IFN-γ, cells were incubated in medium supplemented with 100 ng/mL of either human or mouse IFN-γ (Peprotech) for 48 h before the collection of cell and sEV samples.
Human CD8 T cells were isolated from human blood mononuclear cells (70025, Stemcell Technologies) using Human CD8 + T cell isolation kit (130-096-495, Miltenyi Biotec) while mice T cells were collected from the spleen of C57BL/6 mice (Jackson Laboratory) and isolated with mouse CD8 + T cell isolation kit (130-096-543, Miltenyi Biotec). Human T cells were cultured in T cell expansion medium (10981, STEMCELL Technologies) with 10 ng/mL of human IL-2 (78036.3, STEMCELL Technologies). Mouse T cells were cultured in RPMI medium supplemented with 10% FBS and 10 ng/mL of mouse IL-2 (130-120-334, Miltenyi Biotec). T cells were activated with Human CD3/CD28/CD2 activation kit (10990, STEMCELL Technologies) at 25 µL/mL or Mouse CD3/CD28 beads (11456D, Thermofisher) at 25 µL/mL at every second passage.
Human CD8 T cells were isolated from human blood mononuclear cells (70025, Stemcell Technologies) using Human CD8 + T cell isolation kit (130-096-495, Miltenyi Biotec) while mice T cells were collected from the spleen of C57BL/6 mice (Jackson Laboratory) and isolated with mouse CD8 + T cell isolation kit (130-096-543, Miltenyi Biotec). Human T cells were cultured in T cell expansion medium (10981, STEMCELL Technologies) with 10 ng/mL of human IL-2 (78036.3, STEMCELL Technologies). Mouse T cells were cultured in RPMI medium supplemented with 10% FBS and 10 ng/mL of mouse IL-2 (130-120-334, Miltenyi Biotec). T cells were activated with Human CD3/CD28/CD2 activation kit (10990, STEMCELL Technologies) at 25 µL/mL or Mouse CD3/CD28 beads (11456D, Thermofisher) at 25 µL/mL at every second passage.
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