rosettesep™ human cd4- cd8-depletion cocktails  (STEMCELL Technologies Inc)

Journal: bioRxiv

Article Title: In vivo CRISPR screens reveal Serpinb9 and Adam2 as regulators of immune therapy response in lung cancer

doi: 10.1101/2022.03.13.484176

Figure Lengend Snippet: a , The effect of Adam2 overexpression in LLC cells on CD8 + OT-I T cell mediated killing. CTRL or Adam2 O/E cells labeled with CFSE were used as targets (T) and cocultured with day 4 activated and expanded OT-I CD8 T-cells (E) at different E:T ratios. Flow cytometry analysis was used to calculate the ratio of killed cells at different E:T ratios. Data presents mean ± s.e.m. of three replicates and analysed by two-sided student’s t-test. b , Tumor growth of transplanted CTLR or Adam2 O/E in B6 mice, untreated (n=5) or treated with OT-I T cells (n=5). c , Volcano plot showing differentially expressed genes between OT-I treated CTLR and Adam2 O/E tumors isolated from B6 mice. d , GSEA plots showing Adipogenesis and Peroxisomes pathways in CTLR tumors versus Adam2 O/E tumors. e and f , Bar graph showing changes of Gene Ontology of Biological Process (e) and Molecular Function (f) between OT-I treated CTLR versus Adam2 O/E tumors.

Article Snippet: DNTs were enriched by depleting CD4 + and CD8 + cells using RosetteSep™ human CD4- and CD8-depletion cocktails (Stemcell Technologies).

Techniques: Over Expression, Labeling, Flow Cytometry, Isolation

Journal: PLoS Pathogens

Article Title: HIV infects astrocytes in vivo and egresses from the brain to the periphery

doi: 10.1371/journal.ppat.1008381

Figure Lengend Snippet: Neonate NSG mice were xenotransplanted with HIV- or HIV+ NHAs as described in and HIV detection outside of the brain was evaluated by Real-time PCR products (HIV DNA, A , or HIV RNA, B ) from brain, cervical lymph node, spleen, splenocytes outgrowth assay and splenocytes outgrowth assay supernatant added to fresh PBMCs analyzed by electrophoresis and human GAPDH. Each column indicates individual animal. HIV- animals are shown to left of ladder, HIV+ animals shown to right for all gels shown. No template control (NTC) is the negative control. M/F indicates sex and number is animal number from that group. ( C ) GFP expression in splenocytes at day 14 day of culturing the cells and ( D ) flow cytometry of cultured splenocytes stained for CD3+/CD4+/GFP; dot blots (left) and cumulative data on right ( p = 0.05, Mann-Whitney U-test). ( E ) Representative image of supernatant from splenocytes cultured for 14 days from neonates from HIV- animal (top) or HIV+ animal (bottom) on fresh PBMCs stimulated with soluble α-CD3 and α-CD28 IL-2. Egress for neonates was analyzed in n = 7 (HIV-) and 12 (HIV+) mice; n = 3 (HIV-) and 4 (HIV+) representative mice are shown here; n = 3 (HIV-) and 5 (HIV+) representative mice are shown here.

Article Snippet: Monocytes were depleted using RosetteSep Human Monocyte Depletion Cocktail and CD4+ T cells by RosetteSep Human CD4+ Depletion Cocktail (STEMCELL Technologies, Cambridge, MA) and were injected IP with 2 × 10 7 of the remaining PBMCs.

Techniques: Real-time Polymerase Chain Reaction, Electrophoresis, Control, Negative Control, Expressing, Flow Cytometry, Cell Culture, Staining, MANN-WHITNEY

Journal: PLoS Pathogens

Article Title: HIV infects astrocytes in vivo and egresses from the brain to the periphery

doi: 10.1371/journal.ppat.1008381

Figure Lengend Snippet: Adult NSG mice were xenotransplanted with HIV- or HIV+ NHAs as described in and HIV detection outside of the brain was evaluated by Real-time PCR products (HIV DNA, A , or HIV RNA, B ) from brain, cervical lymph node, spleen, peripheral lymph node, splenocytes outgrowth assay and splenocytes outgrowth assay supernatant added to fresh PBMCs analyzed by electrophoresis and human GAPDH. Each column indicates individual animal. HIV- animals are shown to left of ladder, HIV+ animals shown to right for all gels shown. No template control (NTC) is the negative control. ( C ) GFP expression in splenocytes at day 14 day of culturing the cells and ( D ) flow cytometry of cultured splenocytes stained for CD3+/CD4+/GFP; dot blots (left) and cumulative data on right ( p = 0.04, Mann-Whitney U-test). ( E ) Representative image of supernatant from splenocytes cultured for 14 days from neonates from HIV- animal (top) or HIV+ animal (bottom) on fresh PBMCs stimulated with soluble α-CD3 and α-CD28 IL-2. Egress was analyzed in n = 7 (HIV-) and 9 (HIV+) mice; n = 3 (HIV-) and 5 (HIV+) representative mice are shown here.

Article Snippet: Monocytes were depleted using RosetteSep Human Monocyte Depletion Cocktail and CD4+ T cells by RosetteSep Human CD4+ Depletion Cocktail (STEMCELL Technologies, Cambridge, MA) and were injected IP with 2 × 10 7 of the remaining PBMCs.

Techniques: Real-time Polymerase Chain Reaction, Electrophoresis, Control, Negative Control, Expressing, Flow Cytometry, Cell Culture, Staining, MANN-WHITNEY

Journal: PLoS Pathogens

Article Title: HIV infects astrocytes in vivo and egresses from the brain to the periphery

doi: 10.1371/journal.ppat.1008381

Figure Lengend Snippet: ( A ) Representative image of neonate hippocampus (HPC) immunostained for human T cells (CD3+, magenta), human astrocytes (huGFAP; red), HIV (green) and Nuclei (DAPI, blue) at week 10 post-xenotransplantation and week 4 reconstitution with huPBMCs. Arrows indicates detection of CD3+/HIV+ cell. Scale bar, 10μm. ( B ) Total PBMCs (HIV+) or monocyte/CD4+ T cell depleted PBMCs analyzed by flow cytometry for CD3+/CD4+ positive T cells (top) or CD14 positive or CD14/CD16 positive monocytes before injection into the adult mouse xenotransplanted with HIV VSVg + NHAs. HIV DNA ( C ) and RNA ( D ) from spleen from animals xenotransplanted with HIV VSVg + NHAs and reconstituted with total PBMCs (HIV+) or PBMCs depleted of monocytes and CD4+ T cells (Mono/CD4). No analysis was performed on DNA as Mono/CD4 group had undetectable HIV DNA. RNA is depicted on the right ( p = 0 . 08 , Mann-Whitney U-test). n = 5–7 per group.

Article Snippet: Monocytes were depleted using RosetteSep Human Monocyte Depletion Cocktail and CD4+ T cells by RosetteSep Human CD4+ Depletion Cocktail (STEMCELL Technologies, Cambridge, MA) and were injected IP with 2 × 10 7 of the remaining PBMCs.

Techniques: Flow Cytometry, Injection, MANN-WHITNEY

Journal: PLoS Pathogens

Article Title: HIV infects astrocytes in vivo and egresses from the brain to the periphery

doi: 10.1371/journal.ppat.1008381

Figure Lengend Snippet: Human post mortem analysis of HIV+ astrocytes. Post mortem analysis from cortical or hippocampal brain regions of HIV- or HIV+ individuals. Laser capture microdissection (LCM) of GFAP+ cells analyzed for Alu-gag HIV by PCR. RNAscope and staining determined GFAP+ co-localization with gag mRNA, nef DNA and p24. Staining for each patient included analysis of 12 different sections with 20–24 fields per section (~240 fields) from an average tissue size of 1.5±0.75x0.91±0.66 cm. M: male; F: Female; N.A.: not applicable; U.D.: undetectable. Anti-retrovirals: ATV, atazanavir (Reyataz); EFV, efavirenz (Sustiva); RTV, ritonavir (Norvir); TFV, PMPA; tenofivir DF (Viread); 3TC, lamivudine (Epivir); D4T, stavudine (Zerit FTV, SQV2 or FTV saquinavir-sgc (Fortovase);SQV, saquinavir (Invirase); DDI, didanosine (Videx); NFV, nelfinavir (Viracept). Representative staining images of data are shown in Fig 9 .

Article Snippet: Monocytes were depleted using RosetteSep Human Monocyte Depletion Cocktail and CD4+ T cells by RosetteSep Human CD4+ Depletion Cocktail (STEMCELL Technologies, Cambridge, MA) and were injected IP with 2 × 10 7 of the remaining PBMCs.

Techniques: Laser Capture Microdissection, RNAscope, Staining, Clinical Proteomics