CD8+ cells were purified from single cell suspensions from spleens of naive mice using anti-CD8 (Ly-2) microbeads (Miltenyi Biotech) according to manufacturer’s protocol and labeled with appropriate CellTrace™ Reagent. The labeled CD8+ T cells were then plated onto round bottom 96-well plates coated with 1 μg/ml anti-CD3 (clone 1454-2C11) and 5 μg/ml anti-CD28 (clone 37N). Purified suppressor cells (i.e. MDSCs or Foxp3+ Tregs) were added in indicated ratios and plates were incubated at 37°C. Percent CD8+ T-cell proliferation was measured by assessing dilution of the CellTrace Dye by flow cytometry after 48–72 hours of culture (LSRII flow cytometer, BD Biosciences). Controls were wells without suppressor cells (Stim+) and wells without suppressor cells and anti-CD3/CD28 antibody (Stim−). Details and specific conditions are provided in SEP.
Anti cd8 ly 2 microbeads
Manufactured by Miltenyi Biotec
Anti-CD8 (Ly-2) microbeads are a laboratory product used for the isolation and enrichment of CD8-positive cells from various cell samples. The microbeads are coated with antibodies specific to the CD8 (Ly-2) surface marker, allowing for the magnetic separation of CD8-expressing cells from a heterogeneous cell population.
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Lab products found in correlation
Lsrii flow cytometer, by BD (2 mentions)
Gallios flow cytometer, by Beckman Coulter (1 mentions)
Facsaria 2, by BD (1 mentions)
Celltrace violet ctv proliferation dye, by Thermo Fisher Scientific (1 mentions)
Dynabeads human t expander cd3 cd28, by Thermo Fisher Scientific (1 mentions)
Cd8 microbead, by Miltenyi Biotec (1 mentions)
Celltrace violet ctv, by Thermo Fisher Scientific (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by BD (1 mentions)
5 protocols using anti cd8 ly 2 microbeads
1
Evaluating CD8+ T-cell Suppression by MDSCs and Tregs
2
Thymocyte Subsets Identification and Sorting
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Thymocytes, splenocytes, and lymph node (LN) cells were suspended in 2% newborn calf serum-balanced salt solution and stained with fluorescent Abs of various combinations. Dead cells were excluded on the basis of low forward-light scatter and propidium iodide staining. Data acquisition and analysis were performed on Gallios flow cytometer (BECKMAN COULTER) or FACS Aria II (BD). For S1P1 staining, thymocytes were stained with fluorochrome-conjugated antibodies for more than 35 min in the dark at 4°C in PBS without serum.
To enrich CD4+ SP thymocytes, CD8− thymocytes were obtained by negative selection using anti-CD8 (Ly-2) MicroBeads (Miltenyi Biotec). The cells were then stained with fluorescently labeled antibodies to CD4, CD8, 6C10, CD69, and Qa-2. CD4 SP thymocytes (Rag2p-EGFP+CD4+CD8−) and its subsets SP1 (6C10+CD69+Qa-2−), SP2 (6C10−CD69+Qa-2−), SP3 (CD69−Qa-2−), and SP4 (CD69−Qa-2+) were sorted to >95% purity with a FACS Aria II (BD Biosciences, San Diego, CA, USA). SP1–3 cells used in intravital imaging were defined as CD4+CD8−Qa-2−.
To enrich CD4+ SP thymocytes, CD8− thymocytes were obtained by negative selection using anti-CD8 (Ly-2) MicroBeads (Miltenyi Biotec). The cells were then stained with fluorescently labeled antibodies to CD4, CD8, 6C10, CD69, and Qa-2. CD4 SP thymocytes (Rag2p-EGFP+CD4+CD8−) and its subsets SP1 (6C10+CD69+Qa-2−), SP2 (6C10−CD69+Qa-2−), SP3 (CD69−Qa-2−), and SP4 (CD69−Qa-2+) were sorted to >95% purity with a FACS Aria II (BD Biosciences, San Diego, CA, USA). SP1–3 cells used in intravital imaging were defined as CD4+CD8−Qa-2−.
3
Suppression Assay with Tumor-Associated Macrophages
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For the suppression assays with TAMs, single-cell suspensions of the tumors were obtained as described above and tumor-isolated macrophages (CD45+CD11b+F4/80hiLy6G–) were sorted using a BD FACSAria at over 90% purity. CD8+ T cells isolated from the spleen of naive mice were purified using anti-CD8 (Ly-2) microbeads (Miltenyi Biotec) according to the manufacturer’s protocol. CD8+ T cells were then stained with CellTrace Violet (CTV) proliferation dye (Thermo Fisher Scientific) and plated in complete RPMI media supplemented with 0.05 M β-mercaptoethanol onto round-bottom 96-well plates (1 × 105 cells per well) and were stimulated with anti-CD3/anti-CD28 microbeads (Dynabeads Mouse T-Expander CD3/CD28, Thermo Fisher Scientific). CD8+ T cells were plated with FACS-isolated TAMs and analyzed for CFSE dilution by flow cytometry. Expansion index was determined using FlowJo v10.
4
Evaluation of Tumor-Infiltrating Regulatory T Cells and Macrophages
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For the suppression assays with tumor-isolated Tregs, B16WT and B16IDO tumors were grown in Foxp3GFP mice and Tregs (CD45+TCRβ+CD4+GFP+) sorted at day 15 after tumor implantation. Tregs were incubated with CellTrace Violet (CTV, Invitrogen)-labeled target CD8+ T cells immunomagnetically purified (CD8 Microbeads, Miltenyi Biotec) from spleens of CD45.1+ C57BL/6J congenic mice. Cultures were stimulated for 48–72 h with 0.5 μg/ml soluble αCD3 Ab and irradiated splenocytes before analyses of target CD8+ T-cell CTV dilution (proliferation) and CD44 upregulation (activation) by flow cytometry. For the suppression assays with TAMs, B16WT and B16IDO tumors were grown in C57BL/6J mice. At day 15 after tumor implantation, tumor-isolated macrophages (CD45+ CD11b+F4/80hiLy6G-) were sorted at over 90% purity. CD8+ cells isolated from the spleen of naïve mice were purified using anti-CD8 (Ly-2) microbeads (Miltenyi Biotech) according to the manufacturer’s protocol. CD8+ T cells were then plated in complete RPMI media supplemented with 0.05 M β-mercaptoethanol onto round bottom 96-well plates (1 × 103 cells per well) and were suboptimally stimulated with αCD3/αCD28 microbeads (Dynabeads Human T-Expander CD3/CD28, ThermoFisher). CD8+ T cells were plated with FACS-sorted TAMs at a ratio of 2:1 (E:T) for 48 h before analysis of PD-1 and CD44 upregulation (activation) by flow cytometry.
5
Assessing MDSC Suppression of CD8+ T Cell Proliferation
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Spleens and lymph nodes from naive mice were isolated and ground through 100-μm filters to generate a single cell suspension. After RBC lysis, CD8+ cells were purified using anti-CD8 (Ly-2) microbeads (Miltenyi Biotech) according to manufacturer's protocol and labeled with 1 mM CFSE (Invitrogen) in pre-warmed PBS for 10 min at 37 °C. The CFSE-labeled CD8+ T cells were then plated in complete RPMI media supplemented with 0.05 M β-mercaptoethanol onto round bottom 96-well plates (1 × 105 cells per well) coated with 1 μg/ml anti-CD3 (clone 1454-2C11) and 5 μg/ml anti-CD28 (clone 37N) antibodies. Purified MDSCs were added in indicated ratios and plates were incubated at 37 °C. After 48 h, cells were harvested and CFSE signal in the gated CD8+ T cells was measured by flow cytometry (LSRII Flow Cytometer, BD Biosciences).
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