easysep human naïve cd4 + t cell isolation kit ii  (STEMCELL Technologies Inc)

Journal: bioRxiv

Article Title: Traitor-virus-guided discovery of novel antiviral factors

doi: 10.1101/2023.11.03.565501

Figure Lengend Snippet: a, Expression of cellular factors targeted by gRNAs selected during passage of HIV-1 in CEM-M7 and SupT1 CCR5 high Cas9 cells with or without the indicated IFNs (1000U/ml). Whole-cell lysates were immunoblotted and stained with antibodies against the indicated proteins. b, Percentage of eGFP positive cells indicating infected CEM-M7 Cas9 cells at 4 days post infection electroporated with either the NT or GRN gRNA and infected with WT NL4-3. Bars represent the mean of infected cells at 2dpi relative to the control (100%) of three independent experiments, ±SEM, *p<0.05, Student’s t-test Welch’s correction, ** p<0.001, ***p<0.0001 In the lower panel a representative WB showing PGRN KO efficiency. c, HEK293T cells were cotransfected with increasing amounts of GRN expression construct and proviral mutants of NL4-3 or CH077 lacking the accessory genes. Each point represents the average of three independent experiments ±SEM. In the lower panel a representative WB indicating expression of Env, p55, p24 and PGRN in virus supernatants or cell lysates. d, HEK293T cells were cotransfected with a luciferase reporter constructs under the control of the HIV-1 LTR and expression constructs for GRN in presence and absence of NL4-3 Tat or a vector control. Bars represent the mean of three independent experiments ±SEM, Student’s t-test Welch’s correction, *p<0.05, ** p<0.001, ***p<0.0001. e, HEK293T cells were cotransfected with different amount of GRN expression plasmid with either NL4-3_eGFP, CH077_eGFP or CH058_eGFP. Early infection was measured with Flow cytometry and bars represent the mean fluorescence intensities (MFI) of eGFP in the eGFP+/GRN+ population relative to vector control (100%). Bars represent the mean of three independent experiments ±SEM, Student’s t-test Welch’s correction, *p<0.05, ** p<0.001, ***p<0.0001. f, Representative WB and quantification of PGRN KO.in primary CD4 + T cells. Bars represent the mean of three independent experiments ±SD, Student’s t-test Welch’s correction, *p<0.05, ** p<0.001, ***p<0.0001. g, CD4 + T cells from 3 to 6 donors were electroporated with either the GRN gRNA or the NT control, infected with the indicated WT HIV-1 strains and infectious virus yields measured from 2 to 6dpi by TZM-bl infection assays. Values represent the mean of three to six experiments normalized to the NT control (100%) ±SEM, Two-way anova Sidak’s multiple comparison, *p<0.05, ** p<0.001, ***p<0.0001.

Article Snippet: CD4 + T cells were negatively isolated using the RosetteSep™ Human CD4 + T Cell Enrichment Cocktail (Stem Cell Technologies # 15061) or the EasySep TM Human Naïve CD4 + T cell Isolation Kit (Stem Cell Technologies #17953) according to the manufacturer’s instructions.

Techniques: Expressing, Staining, Infection, Construct, Virus, Luciferase, Plasmid Preparation, Flow Cytometry, Fluorescence, Comparison

Journal: bioRxiv

Article Title: Traitor-virus-guided discovery of novel antiviral factors

doi: 10.1101/2023.11.03.565501

Figure Lengend Snippet: a, Schematic representation of the experimental workflow to KO target genes in primary CD4 + T cells and infect them with WT HIV-.1. b, Primary data showing the replication of the indicated WT HIV-1 strains in primary CD4 + T cells electroporated with PGRN, CIITA or NT gRNA.

Article Snippet: CD4 + T cells were negatively isolated using the RosetteSep™ Human CD4 + T Cell Enrichment Cocktail (Stem Cell Technologies # 15061) or the EasySep TM Human Naïve CD4 + T cell Isolation Kit (Stem Cell Technologies #17953) according to the manufacturer’s instructions.

Techniques:

Journal: bioRxiv

Article Title: Traitor-virus-guided discovery of novel antiviral factors

doi: 10.1101/2023.11.03.565501

Figure Lengend Snippet: a, HEK293T cells were cotransfected with increasing amount of either CIITA, CC2D1B or CEACAM3 expression constructs with and indicated proviral constructs. Values represent the mean of three independent experiments ±SEM, Student’s t-test Welch’s correction, *p<0.05, ** p<0.001, ***p<0.0001. b-d, CD4 + T cells from 3 to 4 donors were electroporated with either the gRNA targeting CIITA , CC2D1B , CEACAM3 or the NT control, infected with the indicated WT HIV-1 strains and infectious virus yields measured from 2 to 6 dpi by TZM-bl infection assays. Values represent the mean of two to six experiments normalized to the NT control (100%) ±SEM, Two-way anova Sidak’s multiple comparison *p<0.05, ** p<0.001, ***p<0.0001. Examples from primary data are shown in . e, Percentage of p24 antigen in the supernatants of CD4 + T cells from three to four donors electroporated with either the gRNA targeting CC2D1B or NT control at 3 days post infection with VSV-G pseudo-typed Δ env NL4-3 or CH077. Bars represent the mean of the infectious viral yield at two days post-infection relative to the control (100%) of three to four independent experiments, ±SEM, Student’s t-test Welch’s correction, *p<0.05, ** p<0.001, ***p<0.0001 In the lower panel a representative WB showing CC2D1B KO efficiency.

Article Snippet: CD4 + T cells were negatively isolated using the RosetteSep™ Human CD4 + T Cell Enrichment Cocktail (Stem Cell Technologies # 15061) or the EasySep TM Human Naïve CD4 + T cell Isolation Kit (Stem Cell Technologies #17953) according to the manufacturer’s instructions.

Techniques: Expressing, Construct, Infection, Virus, Comparison

Journal: bioRxiv

Article Title: Traitor-virus-guided discovery of novel antiviral factors

doi: 10.1101/2023.11.03.565501

Figure Lengend Snippet: a, HEK293T cells were cotransfected with different amount of CIITA expression plasmid with with either NL4-3_eGFP, CH077_eGFP or CH058_eGFP. Early infection was measured with Flow cytometry and bars represent the mean fluorescence intensities (MFI) of eGFP in the eGFP+/AF647+ population relative to vector control (100%). Bars represent the mean of two independent experiments ±SD. b, Representative Western blot showing Env, p24 and CC2D1B in virus containing supernatants or cell lysates of HEN293Ts cotransfected with increasing amounts of CC2D1B and the indicated proviral constructs. c, d, Quantification the WB in panel (b) of the p24 release (c) and Env processing (d). Values represent the mean of three independent experiments ±SD, Unpaired T-test Welch’s correction, *p<0.05, ** p<0.001, ***p<0.0001. e, Representative western blot and quantification of CIITA, CC2D1B and CEACAM3 expression upon KO.in primary CD4 + T cells. Bars represent the mean of three independent experiments ±SD, Unpaired T-test Welch’s correction, *p<0.05, ** p<0.001, ***p<0.0001. f, CD4 + T cells were electroporated with gRNA targeting CIITA or the NT control, infected with the indicated WT HIV-1 strains and infectious virus yields measured from 2 to 6 dpi by TZM-bl infection assays. Shown are representative results.

Article Snippet: CD4 + T cells were negatively isolated using the RosetteSep™ Human CD4 + T Cell Enrichment Cocktail (Stem Cell Technologies # 15061) or the EasySep TM Human Naïve CD4 + T cell Isolation Kit (Stem Cell Technologies #17953) according to the manufacturer’s instructions.

Techniques: Expressing, Plasmid Preparation, Infection, Flow Cytometry, Fluorescence, Western Blot, Virus, Construct

Journal: bioRxiv

Article Title: Traitor-virus-guided discovery of novel antiviral factors

doi: 10.1101/2023.11.03.565501

Figure Lengend Snippet: a, Effect of RhoA overexpression on indicated proviral constructs. Values represent mean of three independent experiments ±SEM. b, RhoA expression upon KO.in primary CD4 + T cells. Bars represent the mean of three independent experiments ±SD, Unpaired T-test Welch’s correction, *p<0.05, ** p<0.001, ***p<0.0001. c, Effect of RhoA in CD4 + T cells infected with the indicated WT HIV-1 strains. Infectious virus yields measured every day for 5 or 6 days. Values represent the mean of three experiments to the control (100%) ±SEM. d, Percentages of viable cells electroporated as described for panel (b). e, Percentages of primary CD4 + T cells electroporated with either sgRNA targeting RHOA or the NT control and with Propidium iodide (PI) at different cell cycle phases. Bars represent the mean of two independent experiments ±SD. f, Primary representative flow cytometry data showing the gating used in panel (f).

Article Snippet: CD4 + T cells were negatively isolated using the RosetteSep™ Human CD4 + T Cell Enrichment Cocktail (Stem Cell Technologies # 15061) or the EasySep TM Human Naïve CD4 + T cell Isolation Kit (Stem Cell Technologies #17953) according to the manufacturer’s instructions.

Techniques: Over Expression, Construct, Expressing, Infection, Virus, 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: 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: 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