ex vivo cytotoxic potential of CD8+ T cells was measured as previously described (45 (link)). Briefly, EL-4 cells (ATCC) were pulsed with 2 µM OVA257-264 peptide (AnaSpec) or no peptide for 1hr at 37°C. Peptide pulsed cells were stained with CFSE (Life Technologies) and unpulsed cells were stained with CellTrace Violet (Life Technologies) per manufacturers instructions. CD8+ T cells were enriched by negative selection using the EasySep Mouse CD8+ T cell enrichment kit (StemCell Technologies). The number of effector cells were determined by quantification of Kb/OVA+ CD8+ T cells and the appropriate numbers were added and the mixtures were incubated for 5 hrs at 37 °C. Following incubation, cells were stained with a vital exclusion dye (Life Technologies) to exclude dead cells. Fixed cells were acquired on an LSR II flow cytometer (BD Biosciences) and analyzed using FlowJo v9.7.2 (Treestar). Specific lysis was calculated as 100 – [100 × ( % survival / average % survival in absence of effector cells)].
Easysep mouse cd8 t cell enrichment kit
Manufactured by STEMCELL
Sourced in Canada
The EasySep Mouse CD8+ T Cell Enrichment Kit is a laboratory tool used to isolate and enrich CD8+ T cells from mouse splenocytes or peripheral blood mononuclear cells. The kit utilizes magnetic particles and a specialized buffer system to efficiently separate the target cell population.
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Lab products found in correlation
Dnase 1, by Roche (3 mentions)
Mouse tumor dissociation kit, by Miltenyi Biotec (2 mentions)
Ca2 mg2, by Thermo Fisher Scientific (2 mentions)
Anti cd3 and anti cd28 antibodies, by BD (2 mentions)
Collagenase a, by Merck Group (2 mentions)
Ova257 264 peptide, by AnaSpec (1 mentions)
Celltrace violet, by Thermo Fisher Scientific (1 mentions)
El4 cells, by American Type Culture Collection (1 mentions)
Vital exclusion dye, by Thermo Fisher Scientific (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
Tc20 cell counter, by Bio-Rad (1 mentions)
Easysep mouse cd25 regulatory t cell positive selection kit, by STEMCELL (1 mentions)
Collagenase type 4, by Merck Group (1 mentions)
E450 cell proliferation dye, by Thermo Fisher Scientific (1 mentions)
Vybrant cfda se cell tracer kit, by Thermo Fisher Scientific (1 mentions)
Lsrfortessa, by BD (1 mentions)
Mouse pandc enrichment kit, by Miltenyi Biotec (1 mentions)
24 well flat bottom plates, by Thermo Fisher Scientific (1 mentions)
Cd8 t cell isolation kit 2, by Miltenyi Biotec (1 mentions)
18 protocols using easysep mouse cd8 t cell enrichment kit
1
Cytotoxicity Assay of CD8+ T Cells
2
Isolation and Characterization of CD8 T Cells
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CD8 T cells were isolated from pooled splenocytes of E7TCR269 or OT-1 mice using an EasySep Mouse CD8-T Cell Enrichment Kit (StemCell Technologies) according to manufacturer’s instructions. Cell viability was determined by Bio-Rad TC20 Cell Counter and purity of isolated cells were determined by FACS.
3
Isolation and Enrichment of Tumor-Infiltrating Immune Cells
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Tumor-infiltrating immune cells were isolated as previously described with some modifications [18 (link), 19 (link)]. Briefly, fresh tumor tissues were cut into small pieces and digested at 37°C for at least 20 min in RPMI 1640 supplemented with 0.05% collagenase Type IV (Sigma-Aldrich, St. Louis, MO), 0.002% DNase I (Roche, Basel, Switzerland) and 20% fetal calf serum (FCS, HyClone Laboratories, Logan, UT). Dissociated cells were then filtered through a 150-μm mesh and mononuclear cells were obtained by Ficoll density gradient centrifugation. Mononuclear cells were collected from the interface, washed in PBS, and resuspended in Tris-NH4Cl solution to lyse residual red blood cells. Cells were washed with PBS twice and were subjected to further processing. Intratumoral CD4+CD25+ T cells (Treg-enriched cells) were enriched from intratumoral mononuclear cells using the EasySep™ Mouse CD25 Regulatory T Cell Positive Selection Kit (Stemcell Technologies) following the manufacturer's instructions. Foxp3 staining was conducted to confirm that more than 80% enriched cells were Foxp3+ cells. Intratumoral CD8+ T cells were selected using EasySep™ Mouse CD8+ T Cell Enrichment Kit (Stemcell Technology). Before further experiments, Treg-enriched cells and CD8+ T cells were cultured in RPMI 1640 containing 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin.
4
Adoptive Transfer of Antigen-Specific CD8+ T Cells
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CD8+ T cells were purified from P14 (GP33–41-specific TCR) and TNP4 (NP396–404-specific TCR) transgenic mice using the EasySep mouse CD8+ T-cell enrichment kit (Stemcell Technologies). Purified CD8+ T cells (1.5 × 106) from P14 mice were stained with CFSE (5 μM) (Vybrant CFDA SE Cell Tracer Kit; Molecular Probes), and cells from TNP4 mice (1.5 × 106) with Cell Proliferation Dye e450 (10 μM) (eBioscience). We adoptively transferred 1.5 × 106 CD8+ T cells from P14 and TNP4 mice by IV route into TTR-NP and B6 control mice. The next day, the mice were infected with 200 pfu of LCMV-Arm IP, and they were killed 4 days later.
Alternatively, 1.5 × 106 CD8+ T cells from P14 and TNP4 mice (Stemcell Technologies) were adoptively transferred IV into RAG-NP and RAG control mice. The mice were killed 7 days later. Isolated lymphocytes from the spleen and liver were stained (CD3, CD4, CD8, and 7-AAD), and the proliferation of CFSE and e450-labeled cells was analyzed by flow cytometry on a BD LSRFortessa (BD Biosciences) and using the FlowJo software (Tree Star).
Alternatively, 1.5 × 106 CD8+ T cells from P14 and TNP4 mice (Stemcell Technologies) were adoptively transferred IV into RAG-NP and RAG control mice. The mice were killed 7 days later. Isolated lymphocytes from the spleen and liver were stained (CD3, CD4, CD8, and 7-AAD), and the proliferation of CFSE and e450-labeled cells was analyzed by flow cytometry on a BD LSRFortessa (BD Biosciences) and using the FlowJo software (Tree Star).
5
Isolation of Murine CD8+ T Cells
6
CD8+ T Cell Purification and Adoptive Transfer
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CD8+ T cells were purified from spleens of naïve P14 mice by negative selection using an EasySep Mouse CD8+ T cell enrichment kit (STEMCELL Technology) per the manufacturer's instructions. The purity of each preparation was flow cytometrically determined to be greater than 98%. One day prior to LCMV infection, naïve C57BL/6J mice were seeded intravenously with 5,000 fluorescent protein (CFP, mTFP1, GFP, mOrange, DsRed, or mRFP1) tagged CD8+ P14 T cells.
7
Enrichment and Purification of CD8+ T Cells and DCs
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CD8+ T cells from P14 or WT mice were magnetically sorted from spleens after mechanical dissociation and red blood cell lysis using the EasySep mouse CD8+ T-cell enrichment kit (Stemcell). Purified T cells were maintained overnight in complete medium at a concentration of 1 x 106 cells/ml before starting coculture with DCs. Purity was assessed by flow cytometry and was > 90%.
For DC purification, spleens were mechanically dissociated and treated with 16 mg/ml collagenase and 10 mg/ml DNAse at 37°C for 30 min and then filtered through a 40-µm cell strainer. Negative cell sorting was performed using the mouse PanDC Enrichment kit (Miltenyi Biotec) according to the manufacturer’s instructions. DCs were conserved overnight in PBS, 1% FCS, and 2 mM EDTA at 4°C before use. Purity of the DC cell suspension was assessed by flow cytometry and reached 70% for the transcriptomic experiments.
For DC purification, spleens were mechanically dissociated and treated with 16 mg/ml collagenase and 10 mg/ml DNAse at 37°C for 30 min and then filtered through a 40-µm cell strainer. Negative cell sorting was performed using the mouse PanDC Enrichment kit (Miltenyi Biotec) according to the manufacturer’s instructions. DCs were conserved overnight in PBS, 1% FCS, and 2 mM EDTA at 4°C before use. Purity of the DC cell suspension was assessed by flow cytometry and reached 70% for the transcriptomic experiments.
8
Evaluating CD8+ T Cell Responses in TB Mice
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Lymph nodes (LNs) were obtained from TB mice and digested for 30 min at 37 °C with collagenase A (0.5 mg/ml; Sigma Aldrich), Dnase I (0.2 mg/ml, Roche), diluted in HBSS with Ca2+/Mg2+ and 20mM EDTA (Invitrogen) was added 5 min at room temperature to stop the reaction. CD8 T cells were isolated from LNs of TB mice using EasySep Mouse CD8+ T Cell Enrichment Kit (STEMCELL) and stimulated with anti CD3 and anti CD28 antibodies (BD Biosciences) for 24h and IFNγ was analyzed by ELISpot (Mabtech), accordingly to manufacturer’s instructions. T cell proliferation was evaluated by flow cytometry using CSFE (BioLegend). Antigen specific CD8 T cell response was evaluated in single cell suspension obtained from LNs of TC1 (HPV16 E6/E7 expressing tumor cells) TB mice by flow cytometry using MHC tetramer (H-2Db HPV 16 E7 – RAHYNIVTF), obtained from D. Weiner (Wistar Institute, Philadelphia, USA).
9
Adoptive Transfer of Transgenic T Cells
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Naïve CD45.1+ OTI and CD90.1+ pmel-1 TCR-transgenic CD8+ T cells were purified from secondary lymphoid organs of transgenic mice using the EasySep Mouse CD8+ T-Cell Enrichment Kit (StemCell Technologies, ref 19853). Mice were intravenously injected with 2.0-2.5 × 105 cells in 100 µL of sterile PBS (ThermoFisher Scienfic, ref 10010023).
10
Isolation and Activation of OT-I CD8+ T cells
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Splenic CD8+ T-cells were purified from secondary lymphoid organs of Drd3+/+ or Drd3−/− OT-I mice using the EasySep Mouse CD8+ T-cell Enrichment Kit (StemCell Technologies; cat # 19853). All in vitro experiments were performed using a complete RPMI medium (supplemented with 10% FBS, 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin). CD8+ T-cells (5 × 105 cells/well) were activated with 1 μg/mL of plate-bound anti-CD3 and anti-CD28 Abs on flat bottom 24-well plates (Thermo Scientific) for 24 h, and the culture supernatant was collected for IL-2 quantification by ELISA as previously described [27 (link)]. In other experiments, CD8+ T-cells were initially activated with plate-bound anti-CD3 and anti-CD28 Abs, with or without recombinant IL-2 (50 U/mL), for 48 h. Afterwards, cells were washed with fresh medium and then cultured on flat bottom 24-well plates in the absence of anti-CD3 and anti-CD28 Abs for an additional 48 h. Finally, cells were stimulated or not with 1 μg/mL of OVA257-264 peptide (pOT-I) for 6 h. Brefeldin A (1 μg/mL) was added during the last 4 h.
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