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rosettesep cd4 negative selection kit  (STEMCELL Technologies Inc)

 
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    STEMCELL Technologies Inc rosettesep cd4 negative selection kit
    A) Chronically HIV infected J-Lat 10.6 cells were treated with the LRAs indicated and expression of GFP monitored over time using live cell imaging. Data represent mean (error) of 4 assay replicates of an representative experiment from 3 independent experiments. B) J-Lat 10.6 cells treated as in (A) were monitored for proliferation by phase confluence using live cell imaging. C) Uninfected primary <t>CD4</t> T cells were treated with LRAs or diluent control for 24 hours, supplemented with etoposide (20 μM), and assessed for apoptosis at 72 hours by measurement of caspase 3/7 activity. Data represent mean (SD) of 4 assay replicates from 3 independent experiments. D) Uninfected primary CD4 T cells, as in (C), were treated with LRAs and assessed for cell proliferation by absolute cell counts at 72 hours. E) Activated primary CD4 T cells were infected with HIV IIIB for 48 hours, treated with DMSO or bryostatin-1 (10 ng/ml) followed by anti-Fas antibody treatment (Clone CH11, 0.5–1.0 μg/ml). LIVE/DEAD viability stain and HIV p24 expression were measured by flow cytometry. Representative results as well as mean (SD) of 3 independent experiments are shown. *** p<0.0001; ** p<0.001; * p<0.05.
    Rosettesep Cd4 Negative Selection Kit, supplied by STEMCELL Technologies Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/rosettesep+cd4+-t+cell+negative+selection+kit/rosettesep+human+b+cell+enrichment+cocktail/pmc07595626-143-20-25
    Average 90 stars, based on 1 article reviews
    rosettesep cd4 negative selection kit - by Bioz Stars, 2026-09
    90/100 stars

    Images

    1) Product Images from "Reactivating latent HIV with PKC agonists induces resistance to apoptosis and is associated with phosphorylation and activation of BCL2"

    Article Title: Reactivating latent HIV with PKC agonists induces resistance to apoptosis and is associated with phosphorylation and activation of BCL2

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1008906

    A) Chronically HIV infected J-Lat 10.6 cells were treated with the LRAs indicated and expression of GFP monitored over time using live cell imaging. Data represent mean (error) of 4 assay replicates of an representative experiment from 3 independent experiments. B) J-Lat 10.6 cells treated as in (A) were monitored for proliferation by phase confluence using live cell imaging. C) Uninfected primary CD4 T cells were treated with LRAs or diluent control for 24 hours, supplemented with etoposide (20 μM), and assessed for apoptosis at 72 hours by measurement of caspase 3/7 activity. Data represent mean (SD) of 4 assay replicates from 3 independent experiments. D) Uninfected primary CD4 T cells, as in (C), were treated with LRAs and assessed for cell proliferation by absolute cell counts at 72 hours. E) Activated primary CD4 T cells were infected with HIV IIIB for 48 hours, treated with DMSO or bryostatin-1 (10 ng/ml) followed by anti-Fas antibody treatment (Clone CH11, 0.5–1.0 μg/ml). LIVE/DEAD viability stain and HIV p24 expression were measured by flow cytometry. Representative results as well as mean (SD) of 3 independent experiments are shown. *** p<0.0001; ** p<0.001; * p<0.05.
    Figure Legend Snippet: A) Chronically HIV infected J-Lat 10.6 cells were treated with the LRAs indicated and expression of GFP monitored over time using live cell imaging. Data represent mean (error) of 4 assay replicates of an representative experiment from 3 independent experiments. B) J-Lat 10.6 cells treated as in (A) were monitored for proliferation by phase confluence using live cell imaging. C) Uninfected primary CD4 T cells were treated with LRAs or diluent control for 24 hours, supplemented with etoposide (20 μM), and assessed for apoptosis at 72 hours by measurement of caspase 3/7 activity. Data represent mean (SD) of 4 assay replicates from 3 independent experiments. D) Uninfected primary CD4 T cells, as in (C), were treated with LRAs and assessed for cell proliferation by absolute cell counts at 72 hours. E) Activated primary CD4 T cells were infected with HIV IIIB for 48 hours, treated with DMSO or bryostatin-1 (10 ng/ml) followed by anti-Fas antibody treatment (Clone CH11, 0.5–1.0 μg/ml). LIVE/DEAD viability stain and HIV p24 expression were measured by flow cytometry. Representative results as well as mean (SD) of 3 independent experiments are shown. *** p<0.0001; ** p<0.001; * p<0.05.

    Techniques Used: Infection, Expressing, Live Cell Imaging, Control, Activity Assay, Staining, Flow Cytometry

    A-C) Single-cell RNAseq (scRNA) was performed on primary CD4 T cells from an ART-suppressed, HIV positive donor treated with bryostatin or control for 24 hours. A) tSNE plot of whole transcriptome scRNA sequencing showing global gene expression differences. B) tSNE plot showing BCL2 positive cells (brown) from both the untreated and bryostatin-treated CD4 T cells. C) Violin plots comparing expression of STAT1 and anti-apoptotic BCL2 family proteins between cells treated with bryostatin and untreated cells. D-E) Western blots showing changes: D) in STAT1 levels over 24 hours in CD4 T cells with graph showing changes of STAT1 over time (STAT1/actin ratio across 3 replicates). Significance (p<0.0023) was determined using a linear regression with the slope being significantly non-zero. E) BCL2 expression of bryostatin-treated CD4 T cells over 72 hours. F) Flow cytometry for intracellular BCL2 expression in T cell subsets was performed. Depicted is aggregated data from (n = 3) HIV positive subjects.
    Figure Legend Snippet: A-C) Single-cell RNAseq (scRNA) was performed on primary CD4 T cells from an ART-suppressed, HIV positive donor treated with bryostatin or control for 24 hours. A) tSNE plot of whole transcriptome scRNA sequencing showing global gene expression differences. B) tSNE plot showing BCL2 positive cells (brown) from both the untreated and bryostatin-treated CD4 T cells. C) Violin plots comparing expression of STAT1 and anti-apoptotic BCL2 family proteins between cells treated with bryostatin and untreated cells. D-E) Western blots showing changes: D) in STAT1 levels over 24 hours in CD4 T cells with graph showing changes of STAT1 over time (STAT1/actin ratio across 3 replicates). Significance (p<0.0023) was determined using a linear regression with the slope being significantly non-zero. E) BCL2 expression of bryostatin-treated CD4 T cells over 72 hours. F) Flow cytometry for intracellular BCL2 expression in T cell subsets was performed. Depicted is aggregated data from (n = 3) HIV positive subjects.

    Techniques Used: Control, Sequencing, Gene Expression, Expressing, Western Blot, Flow Cytometry

    Differentially expressed genes in primary CD4 T cells treated with bryostatin or diluent.
    Figure Legend Snippet: Differentially expressed genes in primary CD4 T cells treated with bryostatin or diluent.

    Techniques Used:

    A-C) CD4 T cells treated with 3 different LRAs (A: bryostatin-1; B: prostratin; C: vorinostat) were assayed for phospho-S70-BCL2 and total BCL2 by western blot. D) Cells treated with PKC inhibitors GF109203X (GF) or Sotrastaurin prior to treatment with bryostatin-1 were assessed for phosphorylation of BCL2 S70. E) CD4 T cells were treated with the PKC inhibitor sotrastaurin prior to treatment with bryostatin-1 and etoposide (ETP). Cells were tracked over 3 days for active caspase 3/7. Graphs show mean (SD) area under the curve for each treatment. * p<0.05; ** p<0.001. F-G) CD4 T cells treated with bryostatin-1 over an hour or over 24 hours were probed for phospho-ERK1/2 (F) or phospho-JNK (G). Western blots showed increased ERK1/2 phosphorylation but not JNK phosphorylation. H) CD4 T cells treated with ERK1/2 inhibitor SCH772984 or JNK inhibitor SP600125 (SP) and bryostatin-1 showed loss of BCL2 phosphorylation with SCH772984 but not SP600125.
    Figure Legend Snippet: A-C) CD4 T cells treated with 3 different LRAs (A: bryostatin-1; B: prostratin; C: vorinostat) were assayed for phospho-S70-BCL2 and total BCL2 by western blot. D) Cells treated with PKC inhibitors GF109203X (GF) or Sotrastaurin prior to treatment with bryostatin-1 were assessed for phosphorylation of BCL2 S70. E) CD4 T cells were treated with the PKC inhibitor sotrastaurin prior to treatment with bryostatin-1 and etoposide (ETP). Cells were tracked over 3 days for active caspase 3/7. Graphs show mean (SD) area under the curve for each treatment. * p<0.05; ** p<0.001. F-G) CD4 T cells treated with bryostatin-1 over an hour or over 24 hours were probed for phospho-ERK1/2 (F) or phospho-JNK (G). Western blots showed increased ERK1/2 phosphorylation but not JNK phosphorylation. H) CD4 T cells treated with ERK1/2 inhibitor SCH772984 or JNK inhibitor SP600125 (SP) and bryostatin-1 showed loss of BCL2 phosphorylation with SCH772984 but not SP600125.

    Techniques Used: Western Blot, Phospho-proteomics

    (A) Primary CD4 T cells were transfected with HA-Casp8p41 followed by treatment with bryostatin or vehicle control. Immunoprecipitation (IP) was performed for BCL2 or control, and immunoblot (IB) for HA and BCL2. (B) SPR sensorgrams showing the dose-dependent binding of BCL2 (0–100 nM) to Casp8p41. (C) Purified BCL2ΔTM-His6 was phosphorylated by CDK1/cyclin B in vitro (see ). The mixture was separated by SDS-PAGE, transferred to nitrocellulose, and stained with fast green FCF, or blotted with anti-phospho-Ser70-BCL2 antibody. (D) Affinity curves showing the dose-dependent binding of BCL2 and phospho-BCL2-S70 (0–100 nM) to Casp8p41, as indicated by percentage of total response units (RUs) after background subtraction. (E) Affinities of BCL2 and phospho-BCL2-S70 for Casp8p41 were calculated. P < 0.0106 versus control by unpaired t-test. Error bars, SD of three independent experiments.
    Figure Legend Snippet: (A) Primary CD4 T cells were transfected with HA-Casp8p41 followed by treatment with bryostatin or vehicle control. Immunoprecipitation (IP) was performed for BCL2 or control, and immunoblot (IB) for HA and BCL2. (B) SPR sensorgrams showing the dose-dependent binding of BCL2 (0–100 nM) to Casp8p41. (C) Purified BCL2ΔTM-His6 was phosphorylated by CDK1/cyclin B in vitro (see ). The mixture was separated by SDS-PAGE, transferred to nitrocellulose, and stained with fast green FCF, or blotted with anti-phospho-Ser70-BCL2 antibody. (D) Affinity curves showing the dose-dependent binding of BCL2 and phospho-BCL2-S70 (0–100 nM) to Casp8p41, as indicated by percentage of total response units (RUs) after background subtraction. (E) Affinities of BCL2 and phospho-BCL2-S70 for Casp8p41 were calculated. P < 0.0106 versus control by unpaired t-test. Error bars, SD of three independent experiments.

    Techniques Used: Transfection, Control, Immunoprecipitation, Western Blot, Binding Assay, Purification, In Vitro, SDS Page, Staining

    A-B) Primary CD4 T cells from HIV positive donors were treated ex vivo with a panel of LRAs at physiologic concentrations, and assessed for intracellular BCL2 and HIV-p24 expression by flow cytometry. Representative FACS plots on unstimulated and maximally stimulated cells are in (A). Full data are represented in (B). A ratio paired t test comparing BCL2+ cells and BCL2- cells in all treatment conditions was used to assess statistical significance. C) Potential LRA-induced proliferation or toxicity was assessed in the treated CD4 T cells from (A-B) by measuring ATP content. D-E) Ex vivo CD4 T cells from 4 ART-suppressed, HIV positive donors were FACS sorted by intracellular BCL2 expression (D) and total HIV DNA measured in sorted cells by digital droplet PCR (E). F) PBMCs from healthy donors and HIV-positive persons were stained for T cell subsets using Live/Dead, CD3, CD4, CD8, CD27, and CD45RO. BCL2 expression in T cell subsets from healthy donors and HIV-infected persons were compared. Statistical significance was determined using multiple t tests.
    Figure Legend Snippet: A-B) Primary CD4 T cells from HIV positive donors were treated ex vivo with a panel of LRAs at physiologic concentrations, and assessed for intracellular BCL2 and HIV-p24 expression by flow cytometry. Representative FACS plots on unstimulated and maximally stimulated cells are in (A). Full data are represented in (B). A ratio paired t test comparing BCL2+ cells and BCL2- cells in all treatment conditions was used to assess statistical significance. C) Potential LRA-induced proliferation or toxicity was assessed in the treated CD4 T cells from (A-B) by measuring ATP content. D-E) Ex vivo CD4 T cells from 4 ART-suppressed, HIV positive donors were FACS sorted by intracellular BCL2 expression (D) and total HIV DNA measured in sorted cells by digital droplet PCR (E). F) PBMCs from healthy donors and HIV-positive persons were stained for T cell subsets using Live/Dead, CD3, CD4, CD8, CD27, and CD45RO. BCL2 expression in T cell subsets from healthy donors and HIV-infected persons were compared. Statistical significance was determined using multiple t tests.

    Techniques Used: Ex Vivo, Expressing, Flow Cytometry, Staining, Infection

    Related Articles

    Isolation:

    Article Title: Reactivating latent HIV with PKC agonists induces resistance to apoptosis and is associated with phosphorylation and activation of BCL2
    Article Snippet: .. Primary uninfected PBMCs were isolated from leukocyte reduction system chambers [ ] and CD4 T cells were isolated using the RosetteSep CD4 negative selection kit (Stem Cell Technologies, Vancouver, Canada) per the manufacturer’s protocol. .. Cells were then separated using Ficoll Paque density gradient centrifugation.

    Selection:

    Article Title: Reactivating latent HIV with PKC agonists induces resistance to apoptosis and is associated with phosphorylation and activation of BCL2
    Article Snippet: .. Primary uninfected PBMCs were isolated from leukocyte reduction system chambers [ ] and CD4 T cells were isolated using the RosetteSep CD4 negative selection kit (Stem Cell Technologies, Vancouver, Canada) per the manufacturer’s protocol. .. Cells were then separated using Ficoll Paque density gradient centrifugation.



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