stem cell technology easy sep cd8+ kit  (STEMCELL Technologies Inc)

Journal: PLOS ONE

Article Title: A mechanistically novel peptide agonist of the IL-7 receptor that addresses limitations of IL-7 cytokine therapy

doi: 10.1371/journal.pone.0286834

Figure Lengend Snippet: Naive CD4+ and CD8+ T-cells, were separately isolated by negative selection from PBMCs, and rested overnight prior to compound exposure. (A) Cells were treated with saturating concentrations of IL-7 (10 nM), MDK1472 (1 μM), and MDK-703 (1 μM) for up to 2 hours and scored for pSTAT5 accumulation by ELISA. (B) The dose response for each compound was measured in human naive CD4+ and CD8+ cells, utilizing an exposure time of 15 min (T max ).

Article Snippet: For preparation of lymphocyte subsets, naïve CD4+ T-cells were isolated via two-stage negative selection with EasySep™ Human Naïve CD4+ T-Cell Isolation Kit II (Stemcell Technologies Cat# 17555); and CD8+ T-cells were isolated by negative selection from PBMCs with Stem Cell Technology Easy Sep CD8+ kit, or obtained freshly prepared from Stanford Blood Center.

Techniques: Isolation, Selection, Enzyme-linked Immunosorbent Assay

Journal: PLOS ONE

Article Title: A mechanistically novel peptide agonist of the IL-7 receptor that addresses limitations of IL-7 cytokine therapy

doi: 10.1371/journal.pone.0286834

Figure Lengend Snippet: Frozen PBMCs from 5 healthy donors were rested overnight and treated with 100 nM MDK-703 or 1nM IL-7, or no added compound, and cultured for 30 days in the presence of compounds. On days 3, 7, 16, and 30, cell aliquots were taken and analyzed by flow cytometry for (A) Ki-67 expression and (B) absolute numbers of CD8 + , CD4 + , Treg, and NK cells. Data are shown as mean ± SEM. Flow cytometry gating data shown in .

Article Snippet: For preparation of lymphocyte subsets, naïve CD4+ T-cells were isolated via two-stage negative selection with EasySep™ Human Naïve CD4+ T-Cell Isolation Kit II (Stemcell Technologies Cat# 17555); and CD8+ T-cells were isolated by negative selection from PBMCs with Stem Cell Technology Easy Sep CD8+ kit, or obtained freshly prepared from Stanford Blood Center.

Techniques: Cell Culture, Flow Cytometry, Expressing

Journal: PLOS ONE

Article Title: A mechanistically novel peptide agonist of the IL-7 receptor that addresses limitations of IL-7 cytokine therapy

doi: 10.1371/journal.pone.0286834

Figure Lengend Snippet: Test compounds induce decrease in cell surface IL-7R α. Rested human CD8+ T-cells were treated with saturating concentrations of the test agonists (10 nM IL-7; 1 μM MDK1472 or MDK-703) or the negative control compounds (untreated, 1 μM MDK1169, or MDK-202) for 20 minutes on ice, then incubated at (A) 37°C or (B) 0°C for varying times to monitor uptake of IL-7Rα from the cell surface. Following timed incubations, the samples were stained, fixed, and analyzed by flow cytometry; and data was collected as median fluorescence intensity (MFI), as detailed in Methods. These data were normalized with the blank value (no added compound) set at 100% surface IL-7Rα, and the signal baseline set at 0. The primary flow data (MFI) is shown in .

Article Snippet: For preparation of lymphocyte subsets, naïve CD4+ T-cells were isolated via two-stage negative selection with EasySep™ Human Naïve CD4+ T-Cell Isolation Kit II (Stemcell Technologies Cat# 17555); and CD8+ T-cells were isolated by negative selection from PBMCs with Stem Cell Technology Easy Sep CD8+ kit, or obtained freshly prepared from Stanford Blood Center.

Techniques: Negative Control, Incubation, Staining, Flow Cytometry, Fluorescence

Journal: PLOS ONE

Article Title: A mechanistically novel peptide agonist of the IL-7 receptor that addresses limitations of IL-7 cytokine therapy

doi: 10.1371/journal.pone.0286834

Figure Lengend Snippet: Frozen PBMCs from 5 healthy donors were rested overnight and left untreated or treated with 100 nM MDK-703 or 1 nM IL-7 in culture. On days 3, 7, 16, and 30, cell aliquots were taken and analyzed by flow cytometry of CD8+ T naïve and memory populations. (A) Schematic of the putative differentiation pathway of the memory T cell compartment. (B) differential gating of naïve and early memory (Tscm) subpopulations. (C) Cell counts of treated and untreated memory subpopulations over time. (D) Stacked bar representation of changes in total memory T cell subpopulations over time. (E) Comparison of CD8+ Tscm expansion on day 16 and day 30 of culture with MDK-703 or IL-7. Data are shown as mean ± SEM. Detailed gating information is provided in .

Article Snippet: For preparation of lymphocyte subsets, naïve CD4+ T-cells were isolated via two-stage negative selection with EasySep™ Human Naïve CD4+ T-Cell Isolation Kit II (Stemcell Technologies Cat# 17555); and CD8+ T-cells were isolated by negative selection from PBMCs with Stem Cell Technology Easy Sep CD8+ kit, or obtained freshly prepared from Stanford Blood Center.

Techniques: Flow Cytometry, Comparison

Journal: PLOS ONE

Article Title: A mechanistically novel peptide agonist of the IL-7 receptor that addresses limitations of IL-7 cytokine therapy

doi: 10.1371/journal.pone.0286834

Figure Lengend Snippet: NSG mice (n = 10 per treatment) engrafted with human CD34+ cells from two donors (5 mice/donor) were dosed once intravenously with 1 mg/kg Fc (gray bars) or MDK-703 (blue bars), and peripheral blood (B-D) and spleen (E-G) were collected and analyzed at the indicated times by flow cytometry. (A) Diagram of the experimental plan. (B) Frequencies of Ki-67+ in CD3+, CD4+, CD8+, Treg, and NK cell populations in peripheral blood on day 7. (C) Absolute cell numbers of CD3+, CD4+, CD8+, Treg, and NK cell populations in peripheral blood on day 12. (D) CD8+ T memory subpopulations in peripheral blood on day 12. (E) Absolute cell numbers of CD3+, CD4+, CD8+, Treg, and NK cell populations in the spleen on day 12. (F) CD8+ T memory subpopulations in the spleen on day 12. (G) TCF1 expression in CD8+ T memory subpopulations in the spleen on day 12. Population gates were drawn based on FMO controls. Statistical analysis was done using Student’s T-Test. *p<0.05, **p<0.005, and ***p<0.0005. Detailed gating information is provided in .

Article Snippet: For preparation of lymphocyte subsets, naïve CD4+ T-cells were isolated via two-stage negative selection with EasySep™ Human Naïve CD4+ T-Cell Isolation Kit II (Stemcell Technologies Cat# 17555); and CD8+ T-cells were isolated by negative selection from PBMCs with Stem Cell Technology Easy Sep CD8+ kit, or obtained freshly prepared from Stanford Blood Center.

Techniques: Flow Cytometry, Expressing

Journal: PLOS ONE

Article Title: A mechanistically novel peptide agonist of the IL-7 receptor that addresses limitations of IL-7 cytokine therapy

doi: 10.1371/journal.pone.0286834

Figure Lengend Snippet: (A) PK of MDK-703: Animals (n = 3) were administered a single dose of 1 mg/kg MDK-703 via IV, SC, or IM. The serum concentration of MDK-703 at the indicated time points was determined by sandwich ELISA. (B and C) PD effect of MDK-703: Three animals were dosed once subcutaneously with 0.3 mg/kg, and blood samples were collected at the indicated time points for absolute lymphocyte counts (B) and immune profiling of CD8, CD4, Treg, and NK cells by flow cytometry (C). Data show mean ±SEM.

Article Snippet: For preparation of lymphocyte subsets, naïve CD4+ T-cells were isolated via two-stage negative selection with EasySep™ Human Naïve CD4+ T-Cell Isolation Kit II (Stemcell Technologies Cat# 17555); and CD8+ T-cells were isolated by negative selection from PBMCs with Stem Cell Technology Easy Sep CD8+ kit, or obtained freshly prepared from Stanford Blood Center.

Techniques: Concentration Assay, Sandwich ELISA, Flow Cytometry

Journal: PLOS ONE

Article Title: A mechanistically novel peptide agonist of the IL-7 receptor that addresses limitations of IL-7 cytokine therapy

doi: 10.1371/journal.pone.0286834

Figure Lengend Snippet: Naive CD4+ and CD8+ T-cells, were separately isolated by negative selection from PBMCs, and rested overnight prior to compound exposure. (A) Cells were treated with saturating concentrations of IL-7 (10 nM), MDK1472 (1 μM), and MDK-703 (1 μM) for up to 2 hours and scored for pSTAT5 accumulation by ELISA. (B) The dose response for each compound was measured in human naive CD4+ and CD8+ cells, utilizing an exposure time of 15 min (T max ).

Article Snippet: CD8+ cells isolated by negative selection from PBMCs with Stem Cell Technology Easy Sep CD8+ kit, or obtained freshly prepared from Stanford Blood Center, were rested overnight in RPMI 1640 containing Pen/Strep, L-Glutamine and 20% FBS; then resuspended to 7.0 x10 6 cells/mL.

Techniques: Isolation, Selection, Enzyme-linked Immunosorbent Assay

Journal: PLOS ONE

Article Title: A mechanistically novel peptide agonist of the IL-7 receptor that addresses limitations of IL-7 cytokine therapy

doi: 10.1371/journal.pone.0286834

Figure Lengend Snippet: Frozen PBMCs from 5 healthy donors were rested overnight and treated with 100 nM MDK-703 or 1nM IL-7, or no added compound, and cultured for 30 days in the presence of compounds. On days 3, 7, 16, and 30, cell aliquots were taken and analyzed by flow cytometry for (A) Ki-67 expression and (B) absolute numbers of CD8 + , CD4 + , Treg, and NK cells. Data are shown as mean ± SEM. Flow cytometry gating data shown in .

Article Snippet: CD8+ cells isolated by negative selection from PBMCs with Stem Cell Technology Easy Sep CD8+ kit, or obtained freshly prepared from Stanford Blood Center, were rested overnight in RPMI 1640 containing Pen/Strep, L-Glutamine and 20% FBS; then resuspended to 7.0 x10 6 cells/mL.

Techniques: Cell Culture, Flow Cytometry, Expressing

Journal: PLOS ONE

Article Title: A mechanistically novel peptide agonist of the IL-7 receptor that addresses limitations of IL-7 cytokine therapy

doi: 10.1371/journal.pone.0286834

Figure Lengend Snippet: Test compounds induce decrease in cell surface IL-7R α. Rested human CD8+ T-cells were treated with saturating concentrations of the test agonists (10 nM IL-7; 1 μM MDK1472 or MDK-703) or the negative control compounds (untreated, 1 μM MDK1169, or MDK-202) for 20 minutes on ice, then incubated at (A) 37°C or (B) 0°C for varying times to monitor uptake of IL-7Rα from the cell surface. Following timed incubations, the samples were stained, fixed, and analyzed by flow cytometry; and data was collected as median fluorescence intensity (MFI), as detailed in Methods. These data were normalized with the blank value (no added compound) set at 100% surface IL-7Rα, and the signal baseline set at 0. The primary flow data (MFI) is shown in .

Article Snippet: CD8+ cells isolated by negative selection from PBMCs with Stem Cell Technology Easy Sep CD8+ kit, or obtained freshly prepared from Stanford Blood Center, were rested overnight in RPMI 1640 containing Pen/Strep, L-Glutamine and 20% FBS; then resuspended to 7.0 x10 6 cells/mL.

Techniques: Negative Control, Incubation, Staining, Flow Cytometry, Fluorescence

Journal: PLOS ONE

Article Title: A mechanistically novel peptide agonist of the IL-7 receptor that addresses limitations of IL-7 cytokine therapy

doi: 10.1371/journal.pone.0286834

Figure Lengend Snippet: Frozen PBMCs from 5 healthy donors were rested overnight and left untreated or treated with 100 nM MDK-703 or 1 nM IL-7 in culture. On days 3, 7, 16, and 30, cell aliquots were taken and analyzed by flow cytometry of CD8+ T naïve and memory populations. (A) Schematic of the putative differentiation pathway of the memory T cell compartment. (B) differential gating of naïve and early memory (Tscm) subpopulations. (C) Cell counts of treated and untreated memory subpopulations over time. (D) Stacked bar representation of changes in total memory T cell subpopulations over time. (E) Comparison of CD8+ Tscm expansion on day 16 and day 30 of culture with MDK-703 or IL-7. Data are shown as mean ± SEM. Detailed gating information is provided in .

Article Snippet: CD8+ cells isolated by negative selection from PBMCs with Stem Cell Technology Easy Sep CD8+ kit, or obtained freshly prepared from Stanford Blood Center, were rested overnight in RPMI 1640 containing Pen/Strep, L-Glutamine and 20% FBS; then resuspended to 7.0 x10 6 cells/mL.

Techniques: Flow Cytometry, Comparison

Journal: PLOS ONE

Article Title: A mechanistically novel peptide agonist of the IL-7 receptor that addresses limitations of IL-7 cytokine therapy

doi: 10.1371/journal.pone.0286834

Figure Lengend Snippet: NSG mice (n = 10 per treatment) engrafted with human CD34+ cells from two donors (5 mice/donor) were dosed once intravenously with 1 mg/kg Fc (gray bars) or MDK-703 (blue bars), and peripheral blood (B-D) and spleen (E-G) were collected and analyzed at the indicated times by flow cytometry. (A) Diagram of the experimental plan. (B) Frequencies of Ki-67+ in CD3+, CD4+, CD8+, Treg, and NK cell populations in peripheral blood on day 7. (C) Absolute cell numbers of CD3+, CD4+, CD8+, Treg, and NK cell populations in peripheral blood on day 12. (D) CD8+ T memory subpopulations in peripheral blood on day 12. (E) Absolute cell numbers of CD3+, CD4+, CD8+, Treg, and NK cell populations in the spleen on day 12. (F) CD8+ T memory subpopulations in the spleen on day 12. (G) TCF1 expression in CD8+ T memory subpopulations in the spleen on day 12. Population gates were drawn based on FMO controls. Statistical analysis was done using Student’s T-Test. *p<0.05, **p<0.005, and ***p<0.0005. Detailed gating information is provided in .

Article Snippet: CD8+ cells isolated by negative selection from PBMCs with Stem Cell Technology Easy Sep CD8+ kit, or obtained freshly prepared from Stanford Blood Center, were rested overnight in RPMI 1640 containing Pen/Strep, L-Glutamine and 20% FBS; then resuspended to 7.0 x10 6 cells/mL.

Techniques: Flow Cytometry, Expressing

Journal: PLOS ONE

Article Title: A mechanistically novel peptide agonist of the IL-7 receptor that addresses limitations of IL-7 cytokine therapy

doi: 10.1371/journal.pone.0286834

Figure Lengend Snippet: (A) PK of MDK-703: Animals (n = 3) were administered a single dose of 1 mg/kg MDK-703 via IV, SC, or IM. The serum concentration of MDK-703 at the indicated time points was determined by sandwich ELISA. (B and C) PD effect of MDK-703: Three animals were dosed once subcutaneously with 0.3 mg/kg, and blood samples were collected at the indicated time points for absolute lymphocyte counts (B) and immune profiling of CD8, CD4, Treg, and NK cells by flow cytometry (C). Data show mean ±SEM.

Article Snippet: CD8+ cells isolated by negative selection from PBMCs with Stem Cell Technology Easy Sep CD8+ kit, or obtained freshly prepared from Stanford Blood Center, were rested overnight in RPMI 1640 containing Pen/Strep, L-Glutamine and 20% FBS; then resuspended to 7.0 x10 6 cells/mL.

Techniques: Concentration Assay, Sandwich ELISA, Flow Cytometry