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Jackson Immuno flow cytometric analysis
Electroporation of mRNA encoding anti-EpCAM CAR in human T cells and cytotoxicity of the CAR-modified T cells ( A ) Schematic diagrams of mRNA CAR constructs used in this study. ( B ) Flow <t>cytometric</t> analysis to examine anti-EpCAM CAR expression on T cells after electroporation. The cells were collected 24 hours after electroporation for analysis. ( C ) Western blot analysis using a CD3ζ-specific antibody confirms the CAR expression in T cells. The cells were collected 24 hours after electroporation for analysis. The endogenous CD3ζ was stained as an internal loading control. ( D ) In vitro cell lysis. Delfia EuTDA cytotoxicity assay (3 hours EuTDA culturing) was used to assess the cytotoxicity of anti-EpCAM RNA CAR-modified T cells against EpCAM-positive human cancer cell lines HRT-18G and SW620, as well as EpCAM-negative ovarian cancer cell line PA-1. mGFP RNA CAR-modified T cells were included as a control. Mean ± SD of three validation runs is represented. *** p
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1) Product Images from "Intraperitoneal immunotherapy with T cells stably and transiently expressing anti-EpCAM CAR in xenograft models of peritoneal carcinomatosis"

Article Title: Intraperitoneal immunotherapy with T cells stably and transiently expressing anti-EpCAM CAR in xenograft models of peritoneal carcinomatosis

Journal: Oncotarget

doi: 10.18632/oncotarget.14592

Electroporation of mRNA encoding anti-EpCAM CAR in human T cells and cytotoxicity of the CAR-modified T cells ( A ) Schematic diagrams of mRNA CAR constructs used in this study. ( B ) Flow cytometric analysis to examine anti-EpCAM CAR expression on T cells after electroporation. The cells were collected 24 hours after electroporation for analysis. ( C ) Western blot analysis using a CD3ζ-specific antibody confirms the CAR expression in T cells. The cells were collected 24 hours after electroporation for analysis. The endogenous CD3ζ was stained as an internal loading control. ( D ) In vitro cell lysis. Delfia EuTDA cytotoxicity assay (3 hours EuTDA culturing) was used to assess the cytotoxicity of anti-EpCAM RNA CAR-modified T cells against EpCAM-positive human cancer cell lines HRT-18G and SW620, as well as EpCAM-negative ovarian cancer cell line PA-1. mGFP RNA CAR-modified T cells were included as a control. Mean ± SD of three validation runs is represented. *** p
Figure Legend Snippet: Electroporation of mRNA encoding anti-EpCAM CAR in human T cells and cytotoxicity of the CAR-modified T cells ( A ) Schematic diagrams of mRNA CAR constructs used in this study. ( B ) Flow cytometric analysis to examine anti-EpCAM CAR expression on T cells after electroporation. The cells were collected 24 hours after electroporation for analysis. ( C ) Western blot analysis using a CD3ζ-specific antibody confirms the CAR expression in T cells. The cells were collected 24 hours after electroporation for analysis. The endogenous CD3ζ was stained as an internal loading control. ( D ) In vitro cell lysis. Delfia EuTDA cytotoxicity assay (3 hours EuTDA culturing) was used to assess the cytotoxicity of anti-EpCAM RNA CAR-modified T cells against EpCAM-positive human cancer cell lines HRT-18G and SW620, as well as EpCAM-negative ovarian cancer cell line PA-1. mGFP RNA CAR-modified T cells were included as a control. Mean ± SD of three validation runs is represented. *** p

Techniques Used: Electroporation, Modification, Construct, Flow Cytometry, Expressing, Western Blot, Staining, In Vitro, Lysis, Cytotoxicity Assay

Generation and expansion of EpCAM-specific CART cells ( A ) Schematics of lentiviral vectors used in the study. The scFv region that recognizes EpCAM was derived from 4D5MOC-B humanized mAb. Anti-EpCAM CAR contains the CD28 and 4-1BB co-stimulatory domains and the CD3zeta T cell activation domain. mGFP CAR control vector was constructed using the GFP sequence instead of EpCAM-specific scFv. ( B ) Schematic illustration of EpCAM-specific CART cell generation and expansion. ( C ) Flow cytometric analysis to detect the surface expression of anti-EpCAM CAR on the genetically modified T cells before and after 21 days of co-culturing with K562A-EpCAM aAPCs using anti-mouse IgG Fab and anti-human CD3 antibodies. Representative FACS plots are shown. ( D ) Increase in the number of CD3+CAR+ T cells over the co-culturing with K562A-EpCAM cells. The number of initially seeded T cells was 2E4 per well. Results from five different PBMC samples are shown. ( E ) Western blot analysis using a CD3ζ-specific antibody confirms the CAR expression in modified T cells 21 days after co-culture with K562A-EpCAM cells. The endogenous CD3ζ was stained as an internal loading control. Lane 1: unmodified T cells; Lane 2: T cells transduced with mGFP CAR lentiviral vectors; Lane 3: T cells transduced with anti-EpCAM CAR lentiviral vectors. ( F ) Phenotyping of the propagated EpCAM-specific CART cells. T cells collected after 21 days of co-culture with K562A-EpCAM cells were assessed by flow cytometry. T cells were gated for the presence of naive (T N , CCR7+CD45RA+), central memory (T CM , CCR7+CD45RA-), effector memory (T EM , CCR7-CD45RA-), and terminally differentiated effector T cells (T EFF , CCR7-CD45RA+). Results from five different PBMC samples are shown.
Figure Legend Snippet: Generation and expansion of EpCAM-specific CART cells ( A ) Schematics of lentiviral vectors used in the study. The scFv region that recognizes EpCAM was derived from 4D5MOC-B humanized mAb. Anti-EpCAM CAR contains the CD28 and 4-1BB co-stimulatory domains and the CD3zeta T cell activation domain. mGFP CAR control vector was constructed using the GFP sequence instead of EpCAM-specific scFv. ( B ) Schematic illustration of EpCAM-specific CART cell generation and expansion. ( C ) Flow cytometric analysis to detect the surface expression of anti-EpCAM CAR on the genetically modified T cells before and after 21 days of co-culturing with K562A-EpCAM aAPCs using anti-mouse IgG Fab and anti-human CD3 antibodies. Representative FACS plots are shown. ( D ) Increase in the number of CD3+CAR+ T cells over the co-culturing with K562A-EpCAM cells. The number of initially seeded T cells was 2E4 per well. Results from five different PBMC samples are shown. ( E ) Western blot analysis using a CD3ζ-specific antibody confirms the CAR expression in modified T cells 21 days after co-culture with K562A-EpCAM cells. The endogenous CD3ζ was stained as an internal loading control. Lane 1: unmodified T cells; Lane 2: T cells transduced with mGFP CAR lentiviral vectors; Lane 3: T cells transduced with anti-EpCAM CAR lentiviral vectors. ( F ) Phenotyping of the propagated EpCAM-specific CART cells. T cells collected after 21 days of co-culture with K562A-EpCAM cells were assessed by flow cytometry. T cells were gated for the presence of naive (T N , CCR7+CD45RA+), central memory (T CM , CCR7+CD45RA-), effector memory (T EM , CCR7-CD45RA-), and terminally differentiated effector T cells (T EFF , CCR7-CD45RA+). Results from five different PBMC samples are shown.

Techniques Used: Derivative Assay, Activation Assay, Plasmid Preparation, Construct, Sequencing, Flow Cytometry, Expressing, Genetically Modified, FACS, Western Blot, Modification, Co-Culture Assay, Staining, Transduction, Cytometry

In vitro cell lysis of EpCAM-positive tumour cells with T cells genetically modified by a lentiviral anti-EpCAM CAR vector ( A ) EpCAM expression on ovarian cancer cells as demonstrated by flow cytometric analysis. Three EpCAM-positive human epithelial ovarian cancer cell lines (CAOV3, SW626, and SKOV3-luc) and one EpCAM-negative human ovarian cancer cell line (PA-1) were analysed. ( B ) % cytotoxicity. Delfia EuTDA cytotoxicity assay (3 hours EuTDA culturing) was used to assess the cytotoxicity of anti-EpCAM CAR-expressing T cells against EpCAM-positive ovarian cancer cell lines. Specific cell lysis was demonstrated by including EpCAM-negative PA-1 cells and the use of mGFP CAR. Mean ± SD of three validation runs is represented.
Figure Legend Snippet: In vitro cell lysis of EpCAM-positive tumour cells with T cells genetically modified by a lentiviral anti-EpCAM CAR vector ( A ) EpCAM expression on ovarian cancer cells as demonstrated by flow cytometric analysis. Three EpCAM-positive human epithelial ovarian cancer cell lines (CAOV3, SW626, and SKOV3-luc) and one EpCAM-negative human ovarian cancer cell line (PA-1) were analysed. ( B ) % cytotoxicity. Delfia EuTDA cytotoxicity assay (3 hours EuTDA culturing) was used to assess the cytotoxicity of anti-EpCAM CAR-expressing T cells against EpCAM-positive ovarian cancer cell lines. Specific cell lysis was demonstrated by including EpCAM-negative PA-1 cells and the use of mGFP CAR. Mean ± SD of three validation runs is represented.

Techniques Used: In Vitro, Lysis, Genetically Modified, Plasmid Preparation, Expressing, Flow Cytometry, Cytotoxicity Assay

Preparation of a K562 aAPC line for expansion of anti-EpCAM CAR-expressing T cells ( A ) Schematic diagrams of plasmid vectors used for the generation of the aAPC cell line. The costimulatory molecule vector (top) was used for transfection of wild-type K562 cells to generate puromycin-resistant K562 cells expressing CD64, CD137L and CD86 (K562A). K562A cells were further modified by co-transfection with the ZFN vector and EpCAM DNA donor vector (bottom) for AAVS1 locus-specific gene insertion to generate puromycin- and neomycin-resistant aAPCs expressing EpCAM (K562A-EpCAM). ( B ) PCR genome typing to demonstrate the AAVS1 locus-specific gene insertion of the EpCAM gene, as indicated by the presence of one single 1.5-kb band. ( C ) Phenotype analysis of K562A-EpCAM cells. Flow cytometric analysis demonstrates the surface expression of CD64, CD86, CD137L, and EpCAM. In the panels for CD64, CD86, and CD137L, left curves: isotype controls; right curves: antibodies. In the panel for EpCAM, left curve: K562 parental cells; right curve: K562A-EpCAM cells.
Figure Legend Snippet: Preparation of a K562 aAPC line for expansion of anti-EpCAM CAR-expressing T cells ( A ) Schematic diagrams of plasmid vectors used for the generation of the aAPC cell line. The costimulatory molecule vector (top) was used for transfection of wild-type K562 cells to generate puromycin-resistant K562 cells expressing CD64, CD137L and CD86 (K562A). K562A cells were further modified by co-transfection with the ZFN vector and EpCAM DNA donor vector (bottom) for AAVS1 locus-specific gene insertion to generate puromycin- and neomycin-resistant aAPCs expressing EpCAM (K562A-EpCAM). ( B ) PCR genome typing to demonstrate the AAVS1 locus-specific gene insertion of the EpCAM gene, as indicated by the presence of one single 1.5-kb band. ( C ) Phenotype analysis of K562A-EpCAM cells. Flow cytometric analysis demonstrates the surface expression of CD64, CD86, CD137L, and EpCAM. In the panels for CD64, CD86, and CD137L, left curves: isotype controls; right curves: antibodies. In the panel for EpCAM, left curve: K562 parental cells; right curve: K562A-EpCAM cells.

Techniques Used: Expressing, Plasmid Preparation, Transfection, Modification, Cotransfection, Polymerase Chain Reaction, Flow Cytometry

2) Product Images from "Immune targeting of fibroblast activation protein triggers recognition of multipotent bone marrow stromal cells and cachexia"

Article Title: Immune targeting of fibroblast activation protein triggers recognition of multipotent bone marrow stromal cells and cachexia

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20130110

IHC staining for FAP in various human tumors, and design and in vitro activity of FAP-reactive CARs. Representative IHC staining for FAP in human melanoma (A), colorectal (B), pancreatic (C), and breast (D) adenocarcinomas. Isotype stains were negative (not depicted). Bars: 400 µm (A); 200 µm (B–D). Schematic of the FAP-reactive CAR constructs FAP5-CAR (E) and Sibro-CAR (F). LS, GM-CSFR leader sequence; V H and V L , variable heavy and light chains; L, 218 linker; CD8, transmembrane domain; CD28, 4-1BB, and CD3-ζ, intracellular signaling domains; m, murine; h, human. Both constructs were cloned into the MSGV1 retroviral vector. Retrovirus containing FAP5-CAR or Sibro-CAR constructs were generated and used to transduce mouse and human T cells, respectively, and flow cytometry was used to assess transduction efficiency at day 2 after transduction for FAP5-CAR (G) and day 8–10 after transduction for Sibro-CAR (H). Solid line is isotype control and filled histogram is FAP5 or Sibrotuzumab stained. Day 5-stimulated untransduced (UnTd) and FAP5-CAR–transduced (Td) mouse T cells were assessed for reactivity against plate-bound BSA, α-CD3 mAb, and recombinant human FAP (r-huFAP), and against HEK293 cell lines expressing or not expressing FAP. After an overnight stimulation, supernatants were assessed for IFN-γ with an IFN-γ ELISA (I), and cells were further assessed for cell surface CD107a expression, and production of IFN-γ and TNF by ICS (J). For ICS, cells are gated on FAP5-CAR Td cells. Day ∼14-stimulated UnTd or Sibro-CAR Td human T cells were assessed for in vitro reactivity as described for mouse. IFN-γ ELISA (K), and ICS results gated on Sibro-CAR Td T cells (L) are shown. Mean ± SD. All results are representative of at least three independent experiments.
Figure Legend Snippet: IHC staining for FAP in various human tumors, and design and in vitro activity of FAP-reactive CARs. Representative IHC staining for FAP in human melanoma (A), colorectal (B), pancreatic (C), and breast (D) adenocarcinomas. Isotype stains were negative (not depicted). Bars: 400 µm (A); 200 µm (B–D). Schematic of the FAP-reactive CAR constructs FAP5-CAR (E) and Sibro-CAR (F). LS, GM-CSFR leader sequence; V H and V L , variable heavy and light chains; L, 218 linker; CD8, transmembrane domain; CD28, 4-1BB, and CD3-ζ, intracellular signaling domains; m, murine; h, human. Both constructs were cloned into the MSGV1 retroviral vector. Retrovirus containing FAP5-CAR or Sibro-CAR constructs were generated and used to transduce mouse and human T cells, respectively, and flow cytometry was used to assess transduction efficiency at day 2 after transduction for FAP5-CAR (G) and day 8–10 after transduction for Sibro-CAR (H). Solid line is isotype control and filled histogram is FAP5 or Sibrotuzumab stained. Day 5-stimulated untransduced (UnTd) and FAP5-CAR–transduced (Td) mouse T cells were assessed for reactivity against plate-bound BSA, α-CD3 mAb, and recombinant human FAP (r-huFAP), and against HEK293 cell lines expressing or not expressing FAP. After an overnight stimulation, supernatants were assessed for IFN-γ with an IFN-γ ELISA (I), and cells were further assessed for cell surface CD107a expression, and production of IFN-γ and TNF by ICS (J). For ICS, cells are gated on FAP5-CAR Td cells. Day ∼14-stimulated UnTd or Sibro-CAR Td human T cells were assessed for in vitro reactivity as described for mouse. IFN-γ ELISA (K), and ICS results gated on Sibro-CAR Td T cells (L) are shown. Mean ± SD. All results are representative of at least three independent experiments.

Techniques Used: Immunohistochemistry, Staining, In Vitro, Activity Assay, Construct, Sequencing, Clone Assay, Plasmid Preparation, Generated, Transduction, Flow Cytometry, Cytometry, Recombinant, Expressing, Enzyme-linked Immunosorbent Assay

3) Product Images from "Neuromyelitis optica study model based on chronic infusion of autoantibodies in rat cerebrospinal fluid"

Article Title: Neuromyelitis optica study model based on chronic infusion of autoantibodies in rat cerebrospinal fluid

Journal: Journal of Neuroinflammation

doi: 10.1186/s12974-016-0577-8

Axonal damage and loss in the spinal cord and optic nerve of the NMO-rat. a Axon injury detected in the NMO-rat compared to the Control-rat (rats infused with IgG AQP4+ 2 and IgG Control 2, D7) using neurofilament immunostaining: reduced number of axons detected as NF-M-positive spots in the white matter (WM); fragmentation and reduced axon thickness in the gray matter (GM). b Classification (10–20 to 100–140 μm 2 , ImageJ) and quantification (mean by field) of NF-M-positive spots in the spinal cord of the NMO-rats ( n = 6) compared to the Control-rats ( n = 6): loss of axons with 60–140 μm 2 sections in the NMO-rats (in cart: evaluation of the total axon number, p = 0.03). c Co-detection of myelin alteration (MBP in green ) and axonal loss (neurofilament NF-M subtype in red ) in the spinal cord of the NMO-rat compared to the Control-rat. d Increased expression of the NF-H phosphorylated form, a marker of axon injury, detected by Western blot (pNF-H/NF-H ratio; p = 0.04). e Axon fragmentation and loss in the optic nerve of the NMO-rats compared to the Control-rat detected by NF-M immunostaining. Scale bars = 20 μm
Figure Legend Snippet: Axonal damage and loss in the spinal cord and optic nerve of the NMO-rat. a Axon injury detected in the NMO-rat compared to the Control-rat (rats infused with IgG AQP4+ 2 and IgG Control 2, D7) using neurofilament immunostaining: reduced number of axons detected as NF-M-positive spots in the white matter (WM); fragmentation and reduced axon thickness in the gray matter (GM). b Classification (10–20 to 100–140 μm 2 , ImageJ) and quantification (mean by field) of NF-M-positive spots in the spinal cord of the NMO-rats ( n = 6) compared to the Control-rats ( n = 6): loss of axons with 60–140 μm 2 sections in the NMO-rats (in cart: evaluation of the total axon number, p = 0.03). c Co-detection of myelin alteration (MBP in green ) and axonal loss (neurofilament NF-M subtype in red ) in the spinal cord of the NMO-rat compared to the Control-rat. d Increased expression of the NF-H phosphorylated form, a marker of axon injury, detected by Western blot (pNF-H/NF-H ratio; p = 0.04). e Axon fragmentation and loss in the optic nerve of the NMO-rats compared to the Control-rat detected by NF-M immunostaining. Scale bars = 20 μm

Techniques Used: Immunostaining, Expressing, Marker, Western Blot

4) Product Images from "Immune targeting of fibroblast activation protein triggers recognition of multipotent bone marrow stromal cells and cachexia"

Article Title: Immune targeting of fibroblast activation protein triggers recognition of multipotent bone marrow stromal cells and cachexia

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20130110

Murine and human multipotent BMSCs express FAP and are recognized by T cells expressing FAP-reactive CARs. Passage-5 in vitro–expanded murine BMSCs were stained with antibodies specific for Sca-1, PDGFR-α, and FAP, and assessed by flow cytometry (A). “Q” represents quadrant. Solid lines are isotype controls and filled histograms are FAP stained. UnTd or FAP5-CAR Td T cells were cultured overnight with murine BMSCs and supernatants were assessed for IFN-γ by ELISA (B), and cells were further analyzed for expression of CD107a and production of IFN-γ and TNF by ICS (C). Mean ± SD. Data are representative of two independent experiments. Flow cytometric phenotype of in vitro–expanded human BMSCs derived from three different donors (D). BMSCs from D were stained with the FAP-specific monoclonal antibodies Sibrotuzumab (E) and FAP5 (F) and assessed by flow cytometry. Solid lines are isotype or secondary antibody controls and filled histograms are FAP or Sibrotuzumab stained. UnTd or Sibro-CAR Td T cells were cultured overnight with BMSCs and the supernatants assessed for IFN-γ by ELISA (G), and cells were further analyzed for CD107a expression and IFN-γ and TNF production by ICS (H). Mean ± SD. Similar results were seen with two additional T cell donors.
Figure Legend Snippet: Murine and human multipotent BMSCs express FAP and are recognized by T cells expressing FAP-reactive CARs. Passage-5 in vitro–expanded murine BMSCs were stained with antibodies specific for Sca-1, PDGFR-α, and FAP, and assessed by flow cytometry (A). “Q” represents quadrant. Solid lines are isotype controls and filled histograms are FAP stained. UnTd or FAP5-CAR Td T cells were cultured overnight with murine BMSCs and supernatants were assessed for IFN-γ by ELISA (B), and cells were further analyzed for expression of CD107a and production of IFN-γ and TNF by ICS (C). Mean ± SD. Data are representative of two independent experiments. Flow cytometric phenotype of in vitro–expanded human BMSCs derived from three different donors (D). BMSCs from D were stained with the FAP-specific monoclonal antibodies Sibrotuzumab (E) and FAP5 (F) and assessed by flow cytometry. Solid lines are isotype or secondary antibody controls and filled histograms are FAP or Sibrotuzumab stained. UnTd or Sibro-CAR Td T cells were cultured overnight with BMSCs and the supernatants assessed for IFN-γ by ELISA (G), and cells were further analyzed for CD107a expression and IFN-γ and TNF production by ICS (H). Mean ± SD. Similar results were seen with two additional T cell donors.

Techniques Used: Expressing, In Vitro, Staining, Flow Cytometry, Cytometry, Cell Culture, Enzyme-linked Immunosorbent Assay, Derivative Assay

Expression of FAP on freshly isolated murine BMSCs from OS cells. BM (A) and OS cells (B) were isolated from untreated wild-type C57BL/6 mice and stained with antibodies against CD45, TER119, Sca-1, PDGFR-α, and FAP, followed by flow cytometry analysis. CD45 + /TER119 + cells demarcate hematopoietic and erythroid lineage cells (Lin + ). Expression of FAP in various populations of OS cells stained with antibodies specific for Sca-1 and PDGFR-α (B). Irradiated non–tumor-bearing mice were treated with 2 × 10 7 UnTd or FAP5-CAR Td T cells and, 7 d later, OS cells were isolated and analyzed as in B. Expression of FAP on various OS cell populations isolated from mice treated with UnTd (C) or FAP5-CAR Td (D) T cells is shown. (E) Mean fluorescence intensity (MFI) of FAP in the various Sca-1 and PDGFR-α subsets found in OS cells. All data are gated on live, single cells. Data for C–E are further gated on Lin − (CD45 − /TER119 − ) cells. Solid lines are isotype controls and filled histograms are FAP5 stained. All data are representative of at least two independent experiments.
Figure Legend Snippet: Expression of FAP on freshly isolated murine BMSCs from OS cells. BM (A) and OS cells (B) were isolated from untreated wild-type C57BL/6 mice and stained with antibodies against CD45, TER119, Sca-1, PDGFR-α, and FAP, followed by flow cytometry analysis. CD45 + /TER119 + cells demarcate hematopoietic and erythroid lineage cells (Lin + ). Expression of FAP in various populations of OS cells stained with antibodies specific for Sca-1 and PDGFR-α (B). Irradiated non–tumor-bearing mice were treated with 2 × 10 7 UnTd or FAP5-CAR Td T cells and, 7 d later, OS cells were isolated and analyzed as in B. Expression of FAP on various OS cell populations isolated from mice treated with UnTd (C) or FAP5-CAR Td (D) T cells is shown. (E) Mean fluorescence intensity (MFI) of FAP in the various Sca-1 and PDGFR-α subsets found in OS cells. All data are gated on live, single cells. Data for C–E are further gated on Lin − (CD45 − /TER119 − ) cells. Solid lines are isotype controls and filled histograms are FAP5 stained. All data are representative of at least two independent experiments.

Techniques Used: Expressing, Isolation, Mouse Assay, Staining, Flow Cytometry, Cytometry, Irradiation, Fluorescence

Bone toxicity and cachexia in mice treated with FAP5-CAR T cells. Irradiated mice were treated with 2 × 10 7 UnTd or FAP5-CAR Td T cells, and 7 d later subjected to a comprehensive necropsy. H E-stained cross section of the femurs from mice treated with UnTd (A) or FAP5-CAR Td (B) T cells. Bars, 400 µm. Femurs and tibias of irradiated and nonirradiated mice that did not undergo adoptive cell transfer (No Tx) or that underwent adoptive transfer with UnTd or FAP5-CAR Td T cells were harvested at day 7 (2 mice pooled per group), and BM (C) and OS (D) cells were isolated and live cells quantitated. Mean ± SD. Data are the average number of cells isolated from the femurs and tibiae of one mouse, and are representative of at least two independent experiments. *, P
Figure Legend Snippet: Bone toxicity and cachexia in mice treated with FAP5-CAR T cells. Irradiated mice were treated with 2 × 10 7 UnTd or FAP5-CAR Td T cells, and 7 d later subjected to a comprehensive necropsy. H E-stained cross section of the femurs from mice treated with UnTd (A) or FAP5-CAR Td (B) T cells. Bars, 400 µm. Femurs and tibias of irradiated and nonirradiated mice that did not undergo adoptive cell transfer (No Tx) or that underwent adoptive transfer with UnTd or FAP5-CAR Td T cells were harvested at day 7 (2 mice pooled per group), and BM (C) and OS (D) cells were isolated and live cells quantitated. Mean ± SD. Data are the average number of cells isolated from the femurs and tibiae of one mouse, and are representative of at least two independent experiments. *, P

Techniques Used: Mouse Assay, Irradiation, Staining, Adoptive Transfer Assay, Isolation

IHC staining for FAP in various human tumors, and design and in vitro activity of FAP-reactive CARs. Representative IHC staining for FAP in human melanoma (A), colorectal (B), pancreatic (C), and breast (D) adenocarcinomas. Isotype stains were negative (not depicted). Bars: 400 µm (A); 200 µm (B–D). Schematic of the FAP-reactive CAR constructs FAP5-CAR (E) and Sibro-CAR (F). LS, GM-CSFR leader sequence; V H and V L , variable heavy and light chains; L, 218 linker; CD8, transmembrane domain; CD28, 4-1BB, and CD3-ζ, intracellular signaling domains; m, murine; h, human. Both constructs were cloned into the MSGV1 retroviral vector. Retrovirus containing FAP5-CAR or Sibro-CAR constructs were generated and used to transduce mouse and human T cells, respectively, and flow cytometry was used to assess transduction efficiency at day 2 after transduction for FAP5-CAR (G) and day 8–10 after transduction for Sibro-CAR (H). Solid line is isotype control and filled histogram is FAP5 or Sibrotuzumab stained. Day 5-stimulated untransduced (UnTd) and FAP5-CAR–transduced (Td) mouse T cells were assessed for reactivity against plate-bound BSA, α-CD3 mAb, and recombinant human FAP (r-huFAP), and against HEK293 cell lines expressing or not expressing FAP. After an overnight stimulation, supernatants were assessed for IFN-γ with an IFN-γ ELISA (I), and cells were further assessed for cell surface CD107a expression, and production of IFN-γ and TNF by ICS (J). For ICS, cells are gated on FAP5-CAR Td cells. Day ∼14-stimulated UnTd or Sibro-CAR Td human T cells were assessed for in vitro reactivity as described for mouse. IFN-γ ELISA (K), and ICS results gated on Sibro-CAR Td T cells (L) are shown. Mean ± SD. All results are representative of at least three independent experiments.
Figure Legend Snippet: IHC staining for FAP in various human tumors, and design and in vitro activity of FAP-reactive CARs. Representative IHC staining for FAP in human melanoma (A), colorectal (B), pancreatic (C), and breast (D) adenocarcinomas. Isotype stains were negative (not depicted). Bars: 400 µm (A); 200 µm (B–D). Schematic of the FAP-reactive CAR constructs FAP5-CAR (E) and Sibro-CAR (F). LS, GM-CSFR leader sequence; V H and V L , variable heavy and light chains; L, 218 linker; CD8, transmembrane domain; CD28, 4-1BB, and CD3-ζ, intracellular signaling domains; m, murine; h, human. Both constructs were cloned into the MSGV1 retroviral vector. Retrovirus containing FAP5-CAR or Sibro-CAR constructs were generated and used to transduce mouse and human T cells, respectively, and flow cytometry was used to assess transduction efficiency at day 2 after transduction for FAP5-CAR (G) and day 8–10 after transduction for Sibro-CAR (H). Solid line is isotype control and filled histogram is FAP5 or Sibrotuzumab stained. Day 5-stimulated untransduced (UnTd) and FAP5-CAR–transduced (Td) mouse T cells were assessed for reactivity against plate-bound BSA, α-CD3 mAb, and recombinant human FAP (r-huFAP), and against HEK293 cell lines expressing or not expressing FAP. After an overnight stimulation, supernatants were assessed for IFN-γ with an IFN-γ ELISA (I), and cells were further assessed for cell surface CD107a expression, and production of IFN-γ and TNF by ICS (J). For ICS, cells are gated on FAP5-CAR Td cells. Day ∼14-stimulated UnTd or Sibro-CAR Td human T cells were assessed for in vitro reactivity as described for mouse. IFN-γ ELISA (K), and ICS results gated on Sibro-CAR Td T cells (L) are shown. Mean ± SD. All results are representative of at least three independent experiments.

Techniques Used: Immunohistochemistry, Staining, In Vitro, Activity Assay, Construct, Sequencing, Clone Assay, Plasmid Preparation, Generated, Transduction, Flow Cytometry, Cytometry, Recombinant, Expressing, Enzyme-linked Immunosorbent Assay

FAP expression in mouse tumors, and in vivo activity of FAP5-CAR–transduced T cells against various murine tumors. In vitro cultured B16, MC38, MC17-51, 4T1, CT26, and Renca murine tumors were assessed for FAP expression by flow cytometry with the FAP-specific antibody FAP5 (A). Solid line is isotype control and filled histogram is FAP5 stained. Results are representative of at least two independent experiments. Established (∼11–16 d) subcutaneously implanted B16 (B), MC38 (C), MC17-51 (D), 4T1 (E), CT26 (F), and Renca (G) tumors were harvested from mice (irradiated before harvest) and assessed for FAP expression by IHC using biotinylated-FAP5 antibody. Bars, 400 µm. Representative of at least two independent experiments. C57BL/6 mice bearing established B16 (H), MC38 (I), MC17-51 (J) tumors, and BALB/c mice bearing established 4T1 (K), CT26 (L), or Renca (M) tumors were left untreated (No Tx) or treated with 10 7 UnTd or 10 7 FAP5-CAR Td T cells, and the perpendicular diameters of the tumors were measured over time. Mean ± SEM. Results are representative of at least two independent experiments for H–J and one experiment for K–M with initially five mice per group. *, P
Figure Legend Snippet: FAP expression in mouse tumors, and in vivo activity of FAP5-CAR–transduced T cells against various murine tumors. In vitro cultured B16, MC38, MC17-51, 4T1, CT26, and Renca murine tumors were assessed for FAP expression by flow cytometry with the FAP-specific antibody FAP5 (A). Solid line is isotype control and filled histogram is FAP5 stained. Results are representative of at least two independent experiments. Established (∼11–16 d) subcutaneously implanted B16 (B), MC38 (C), MC17-51 (D), 4T1 (E), CT26 (F), and Renca (G) tumors were harvested from mice (irradiated before harvest) and assessed for FAP expression by IHC using biotinylated-FAP5 antibody. Bars, 400 µm. Representative of at least two independent experiments. C57BL/6 mice bearing established B16 (H), MC38 (I), MC17-51 (J) tumors, and BALB/c mice bearing established 4T1 (K), CT26 (L), or Renca (M) tumors were left untreated (No Tx) or treated with 10 7 UnTd or 10 7 FAP5-CAR Td T cells, and the perpendicular diameters of the tumors were measured over time. Mean ± SEM. Results are representative of at least two independent experiments for H–J and one experiment for K–M with initially five mice per group. *, P

Techniques Used: Expressing, In Vivo, Activity Assay, In Vitro, Cell Culture, Flow Cytometry, Cytometry, Staining, Mouse Assay, Irradiation, Immunohistochemistry

5) Product Images from "STING couples with PI3K to regulate actin reorganization during BCR activation"

Article Title: STING couples with PI3K to regulate actin reorganization during BCR activation

Journal: Science Advances

doi: 10.1126/sciadv.aax9455

STING is involved in BCR activation. Purified splenic B cells from WT and Sting KO mice were labeled with AF546-mB-Fab-anti-Ig and activated by incubating with either streptavidin or the medium alone (0 min) as a control at 37°C for varying lengths of time. After fixation and permeabilization, samples of WT were stained with antibodies for STING or ER-Tracker Blue-White DPX dye, and samples of KO were stained with ER-Tracker Blue-White DPX dye. Then, samples were analyzed using confocal microscopy. Shown are representative images ( A and D ) and the correlation coefficients between the labeled BCR and STING ( B ) or ER ( E ) quantified using the ZEN 2.3 (blue edition) software. Scale bars, 2.5 μm. Splenic B cells from WT and Sting KO mice were lysed, and then the lysates were probed with antibodies specific for STING ( C ). Enzyme-linked immunosorbent assay (ELISA) quantification of anti-dsDNA Ab in the serum of WT and Sting KO mice ( n = 9). Dots represent individual mice ( F ). Immunofluorescence analysis of nephritic sections. Shown are representative glomeruli ( G and H ). 60× objective; scale bars, 25 μm. Flow cytometry analysis of GCB cells in spleens from aging ( > 5 months) and young (6- to 8-week-old) WT and Sting KO mice. Shown are representative dot plots of aging mice and the average percentages (±SD) and numbers of GCB cells in spleens of WT and Sting KO mice ( I and J ). H E staining of kidney, lung, colon, and liver from WT and Sting KO mice. Shown are representative images and the pathological score from five mice ( K and L ). Each item was scored on a 5-point scale as follows: 0 = minimal damage, 1+ = mild damage, 2+ =moderate damage, 3+ =severe damage, and 4+ =maximal damage. For kidney, scale bar is 20 μm. For lung, colon, and liver, scale bars are 200 μm. * P
Figure Legend Snippet: STING is involved in BCR activation. Purified splenic B cells from WT and Sting KO mice were labeled with AF546-mB-Fab-anti-Ig and activated by incubating with either streptavidin or the medium alone (0 min) as a control at 37°C for varying lengths of time. After fixation and permeabilization, samples of WT were stained with antibodies for STING or ER-Tracker Blue-White DPX dye, and samples of KO were stained with ER-Tracker Blue-White DPX dye. Then, samples were analyzed using confocal microscopy. Shown are representative images ( A and D ) and the correlation coefficients between the labeled BCR and STING ( B ) or ER ( E ) quantified using the ZEN 2.3 (blue edition) software. Scale bars, 2.5 μm. Splenic B cells from WT and Sting KO mice were lysed, and then the lysates were probed with antibodies specific for STING ( C ). Enzyme-linked immunosorbent assay (ELISA) quantification of anti-dsDNA Ab in the serum of WT and Sting KO mice ( n = 9). Dots represent individual mice ( F ). Immunofluorescence analysis of nephritic sections. Shown are representative glomeruli ( G and H ). 60× objective; scale bars, 25 μm. Flow cytometry analysis of GCB cells in spleens from aging ( > 5 months) and young (6- to 8-week-old) WT and Sting KO mice. Shown are representative dot plots of aging mice and the average percentages (±SD) and numbers of GCB cells in spleens of WT and Sting KO mice ( I and J ). H E staining of kidney, lung, colon, and liver from WT and Sting KO mice. Shown are representative images and the pathological score from five mice ( K and L ). Each item was scored on a 5-point scale as follows: 0 = minimal damage, 1+ = mild damage, 2+ =moderate damage, 3+ =severe damage, and 4+ =maximal damage. For kidney, scale bar is 20 μm. For lung, colon, and liver, scale bars are 200 μm. * P

Techniques Used: Activation Assay, Purification, Mouse Assay, Labeling, Staining, Confocal Microscopy, Software, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Flow Cytometry

STING deficiency up-regulates positive BCR signaling. Confocal analysis of splenic B cells from WT and KO mice labeled with AF546-mB-Fab–anti-Ig and then without or with streptavidin to activate. Cells were fixed, permeabilized, and stained with Abs specific for pY, pBtk ( A ), and pCD19 ( I ). Shown are representative images and the correlation coefficients between the labeled BCR and pY/pBtk ( B ) and pCD19 ( J ) from three independent experiments. Scale bars, 2.5 μm. Splenic B cells from WT and Sting KO mice were activated with biotin-conjugated F(ab’) 2 anti-mouse Ig (M + G) plus streptavidin for indicated times. Cell lysates were analyzed using SDS–polyacrylamide gel electrophoresis (PAGE) and Western blot and probed for pY, pBtk, Btk, pSyk, Syk ( C ), and pCD19 ( K ). Total protein or β-actin as controls. Shown are representative blots of three independent experiments and blots’ relative intensity ( D to F and L ). Western blot analysis of the level of pY in purified B cells from PBMCs of the healthy control and STING mutant patient ( G and H ). Flow cytometry analysis of MFI of CD19 in B220 + B cells from WT and Sting KO mice ( M ). * P
Figure Legend Snippet: STING deficiency up-regulates positive BCR signaling. Confocal analysis of splenic B cells from WT and KO mice labeled with AF546-mB-Fab–anti-Ig and then without or with streptavidin to activate. Cells were fixed, permeabilized, and stained with Abs specific for pY, pBtk ( A ), and pCD19 ( I ). Shown are representative images and the correlation coefficients between the labeled BCR and pY/pBtk ( B ) and pCD19 ( J ) from three independent experiments. Scale bars, 2.5 μm. Splenic B cells from WT and Sting KO mice were activated with biotin-conjugated F(ab’) 2 anti-mouse Ig (M + G) plus streptavidin for indicated times. Cell lysates were analyzed using SDS–polyacrylamide gel electrophoresis (PAGE) and Western blot and probed for pY, pBtk, Btk, pSyk, Syk ( C ), and pCD19 ( K ). Total protein or β-actin as controls. Shown are representative blots of three independent experiments and blots’ relative intensity ( D to F and L ). Western blot analysis of the level of pY in purified B cells from PBMCs of the healthy control and STING mutant patient ( G and H ). Flow cytometry analysis of MFI of CD19 in B220 + B cells from WT and Sting KO mice ( M ). * P

Techniques Used: Mouse Assay, Labeling, Staining, Polyacrylamide Gel Electrophoresis, Western Blot, Purification, Mutagenesis, Flow Cytometry

STING deficiency down-regulates the activation of negative BCR signaling molecule, SHIP, and up-regulates the actin polymerization via enhancing the activation of WASP. Confocal analysis of splenic B cells from WT and KO mice labeled with AF546-mB-Fab-anti-Ig and then incubated without or with streptavidin for activation. Cells were fixed, permeabilized, and stained with Abs specific for pSHIP, pWASP, and F-actin ( A and F ). Splenic B cells from WT and Sting KO mice were activated with biotin-conjugated F(ab’) 2 anti-mouse Ig(M + G) plus streptavidin for indicated times. Cell lysates were analyzed using SDS-PAGE and Western blot and probed for pSHIP/SHIP, pSHP1/SHP1 ( C ). Total protein or β-actin was used as controls. Shown are representative images, blots, and the correlation coefficients between the labeled BCR and pSHIP ( B ), pWASP ( G ), as well as the relative intensity of bands from immunoblots ( D and E ) taken from three independent experiments. Splenic B cells were labeled with anti-B220 and stimulated with sAg for indicated times and then stained with phalloidin and antibody specific for pWASP for phos flow cytometry. MFI of pWASP and F-actin in B cells was quantified using FlowJo software ( H and I ). Shown are levels of pWASP and F-actin from one of three independent experiments. Scale bars, 2.5 μm. * P
Figure Legend Snippet: STING deficiency down-regulates the activation of negative BCR signaling molecule, SHIP, and up-regulates the actin polymerization via enhancing the activation of WASP. Confocal analysis of splenic B cells from WT and KO mice labeled with AF546-mB-Fab-anti-Ig and then incubated without or with streptavidin for activation. Cells were fixed, permeabilized, and stained with Abs specific for pSHIP, pWASP, and F-actin ( A and F ). Splenic B cells from WT and Sting KO mice were activated with biotin-conjugated F(ab’) 2 anti-mouse Ig(M + G) plus streptavidin for indicated times. Cell lysates were analyzed using SDS-PAGE and Western blot and probed for pSHIP/SHIP, pSHP1/SHP1 ( C ). Total protein or β-actin was used as controls. Shown are representative images, blots, and the correlation coefficients between the labeled BCR and pSHIP ( B ), pWASP ( G ), as well as the relative intensity of bands from immunoblots ( D and E ) taken from three independent experiments. Splenic B cells were labeled with anti-B220 and stimulated with sAg for indicated times and then stained with phalloidin and antibody specific for pWASP for phos flow cytometry. MFI of pWASP and F-actin in B cells was quantified using FlowJo software ( H and I ). Shown are levels of pWASP and F-actin from one of three independent experiments. Scale bars, 2.5 μm. * P

Techniques Used: Activation Assay, Mouse Assay, Labeling, Incubation, Staining, SDS Page, Western Blot, Flow Cytometry, Software

PI3K inhibition rescues the abnormal accumulation of F-actin in Sting KO B cells. Splenic B cells from WT and Sting KO mice, pretreated with or without PI3K inhibitor, were incubated with AF546-mB-Fab-anti-Ig tethered to lipid bilayers with varying lengths of time and then fixed, permeabilized, and stained with antibodies specific for pY and F-actin. Cells were analyzed using TIRFm. Shown are representative images ( A to D ), the average values of the B cell contact area ( F ), and the MFI of the pY ( G ), BCR ( E ), and F-actin ( H ) in the contact zone. Scale bars, 2.5 μm. Splenic B cells from WT and Sting KO mice were activated with biotin-conjugated F(ab’) 2 anti-mouse Ig(M + G) plus streptavidin for indicated times. Cell lysates were analyzed using SDS-PAGE and Western blot and probed for pPI3K (p85)/PI3K (p85), pAtk/Akt, pS6/S6 ( I ), and pFoxO-1/FoxO-1 ( J ). Total protein or β-actin was used as controls. Shown are representative blots from three independent experiments and blots’ relative intensity ( K to N ). Western blot analysis the level of pPI3K in purified B cells from PBMCs of the healthy control and STING mutant patient ( O and P ). * P
Figure Legend Snippet: PI3K inhibition rescues the abnormal accumulation of F-actin in Sting KO B cells. Splenic B cells from WT and Sting KO mice, pretreated with or without PI3K inhibitor, were incubated with AF546-mB-Fab-anti-Ig tethered to lipid bilayers with varying lengths of time and then fixed, permeabilized, and stained with antibodies specific for pY and F-actin. Cells were analyzed using TIRFm. Shown are representative images ( A to D ), the average values of the B cell contact area ( F ), and the MFI of the pY ( G ), BCR ( E ), and F-actin ( H ) in the contact zone. Scale bars, 2.5 μm. Splenic B cells from WT and Sting KO mice were activated with biotin-conjugated F(ab’) 2 anti-mouse Ig(M + G) plus streptavidin for indicated times. Cell lysates were analyzed using SDS-PAGE and Western blot and probed for pPI3K (p85)/PI3K (p85), pAtk/Akt, pS6/S6 ( I ), and pFoxO-1/FoxO-1 ( J ). Total protein or β-actin was used as controls. Shown are representative blots from three independent experiments and blots’ relative intensity ( K to N ). Western blot analysis the level of pPI3K in purified B cells from PBMCs of the healthy control and STING mutant patient ( O and P ). * P

Techniques Used: Inhibition, Mouse Assay, Incubation, Staining, SDS Page, Western Blot, Purification, Mutagenesis

6) Product Images from "The Bacterial Enzyme IdeS Cleaves the IgG-Type of B Cell Receptor (BCR), Abolishes BCR-Mediated Cell Signaling, and Inhibits Memory B Cell Activation"

Article Title: The Bacterial Enzyme IdeS Cleaves the IgG-Type of B Cell Receptor (BCR), Abolishes BCR-Mediated Cell Signaling, and Inhibits Memory B Cell Activation

Journal: The Journal of Immunology Author Choice

doi: 10.4049/jimmunol.1501929

IdeS cleaves IgG-type, but not IgM-type, of BCR on B cells. ( A ) Flow cytometry analysis of cells stained with biotinylated anti-Fab Ab, followed by streptavidin-allophycocyanin, after treatment of Nu-DUL-1 cells (IgG-type) and Daudi cells (IgM-type) with
Figure Legend Snippet: IdeS cleaves IgG-type, but not IgM-type, of BCR on B cells. ( A ) Flow cytometry analysis of cells stained with biotinylated anti-Fab Ab, followed by streptavidin-allophycocyanin, after treatment of Nu-DUL-1 cells (IgG-type) and Daudi cells (IgM-type) with

Techniques Used: Flow Cytometry, Cytometry, Staining

7) Product Images from "The Bacterial Enzyme IdeS Cleaves the IgG-Type of B Cell Receptor (BCR), Abolishes BCR-Mediated Cell Signaling, and Inhibits Memory B Cell Activation"

Article Title: The Bacterial Enzyme IdeS Cleaves the IgG-Type of B Cell Receptor (BCR), Abolishes BCR-Mediated Cell Signaling, and Inhibits Memory B Cell Activation

Journal: The Journal of Immunology Author Choice

doi: 10.4049/jimmunol.1501929

IdeS cleaves IgG-type, but not IgM-type, of BCR on B cells. ( A ) Flow cytometry analysis of cells stained with biotinylated anti-Fab Ab, followed by streptavidin-allophycocyanin, after treatment of Nu-DUL-1 cells (IgG-type) and Daudi cells (IgM-type) with
Figure Legend Snippet: IdeS cleaves IgG-type, but not IgM-type, of BCR on B cells. ( A ) Flow cytometry analysis of cells stained with biotinylated anti-Fab Ab, followed by streptavidin-allophycocyanin, after treatment of Nu-DUL-1 cells (IgG-type) and Daudi cells (IgM-type) with

Techniques Used: Flow Cytometry, Cytometry, Staining

8) Product Images from "Intraperitoneal immunotherapy with T cells stably and transiently expressing anti-EpCAM CAR in xenograft models of peritoneal carcinomatosis"

Article Title: Intraperitoneal immunotherapy with T cells stably and transiently expressing anti-EpCAM CAR in xenograft models of peritoneal carcinomatosis

Journal: Oncotarget

doi: 10.18632/oncotarget.14592

IFNγ secretion and granzyme B up-regulation triggered by tumor antigen-specific recognition of anti-EpCAM RNA CARs ( A and B ) Increased IFNγ secretion as determined by IFNγ ELISPOT assay. T cells were electroporated with anti-EpCAM RNA CARs and co-cultured with target tumor cells overnight before assay. mGFP RNA CAR-transfected T cells served as a negative control. Results from EpCAM-negative tumor cell lines Raji, IGR1, and PA-1 and EpCAM-positive cancer cell lines SKOV3-Luc and CAOV-3 are shown in ( A ) and results from EpCAM-positive cancer cell lines HCT8, HRT-18G, SW480, SW620 and SW626 in ( B ). ( C ) Granzyme B up-regulation as determined by granzyme B ELISPOT assay. EpCAM-positive tumor cell lines HRT-18G and SKOV3-Luc were tested as described in (A and B) Mean IFNγ or granzyme B spots per 1 × 10 6 T cells ± SD from triplicate cultures are shown in A, B and C.
Figure Legend Snippet: IFNγ secretion and granzyme B up-regulation triggered by tumor antigen-specific recognition of anti-EpCAM RNA CARs ( A and B ) Increased IFNγ secretion as determined by IFNγ ELISPOT assay. T cells were electroporated with anti-EpCAM RNA CARs and co-cultured with target tumor cells overnight before assay. mGFP RNA CAR-transfected T cells served as a negative control. Results from EpCAM-negative tumor cell lines Raji, IGR1, and PA-1 and EpCAM-positive cancer cell lines SKOV3-Luc and CAOV-3 are shown in ( A ) and results from EpCAM-positive cancer cell lines HCT8, HRT-18G, SW480, SW620 and SW626 in ( B ). ( C ) Granzyme B up-regulation as determined by granzyme B ELISPOT assay. EpCAM-positive tumor cell lines HRT-18G and SKOV3-Luc were tested as described in (A and B) Mean IFNγ or granzyme B spots per 1 × 10 6 T cells ± SD from triplicate cultures are shown in A, B and C.

Techniques Used: Enzyme-linked Immunospot, Cell Culture, Transfection, Negative Control

9) Product Images from "The Bacterial Enzyme IdeS Cleaves the IgG-Type of B Cell Receptor (BCR), Abolishes BCR-Mediated Cell Signaling, and Inhibits Memory B Cell Activation"

Article Title: The Bacterial Enzyme IdeS Cleaves the IgG-Type of B Cell Receptor (BCR), Abolishes BCR-Mediated Cell Signaling, and Inhibits Memory B Cell Activation

Journal: The Journal of Immunology Author Choice

doi: 10.4049/jimmunol.1501929

IdeS cleaves IgG-type, but not IgM-type, of BCR on B cells. ( A ) Flow cytometry analysis of cells stained with biotinylated anti-Fab Ab, followed by streptavidin-allophycocyanin, after treatment of Nu-DUL-1 cells (IgG-type) and Daudi cells (IgM-type) with
Figure Legend Snippet: IdeS cleaves IgG-type, but not IgM-type, of BCR on B cells. ( A ) Flow cytometry analysis of cells stained with biotinylated anti-Fab Ab, followed by streptavidin-allophycocyanin, after treatment of Nu-DUL-1 cells (IgG-type) and Daudi cells (IgM-type) with

Techniques Used: Flow Cytometry, Cytometry, Staining

Related Articles

Incubation:

Article Title: STING couples with PI3K to regulate actin reorganization during BCR activation
Article Snippet: .. For Phosflow, primary B cells were incubated with biotin-conjugated F(ab’)2 anti-mouse Ig(M + G) (115-066-068, Jackson ImmunoResearch) plus streptavidin at 37°C for varying lengths of time. .. Cells were fixed with Phosflow Lyse/Fix buffer, permeabilized with Phosflow Perm buffer III (BD Biosciences), and labeled with AF488-phalloidin, anti-pWASP, and PE-PIP2 (Ab01220-10.0, Absolute Antibody).

Article Title: WASP and Mst1 coregulate B-cell development and B-cell receptor signaling
Article Snippet: .. For coimmunoprecipitation, purified splenic B cells (5 × 106 ) were incubated with or without Biotin-conjugated F(ab')2 Anti-Mouse Ig (G+M) (115-066-068; Jackson ImmunoResearch Laboratories) for indicated times in the presence or absence of streptavidin. .. Cells were then lysed with a 150 μL radioimmunoprecipitation assay lysis buffer (Beyotime, P0013B) containing protease inhibitor cocktail (G2006; Servicebio).The cell lysates were incubated with 1 μg of antibody anti-Mst1 or control immunoglobulin G (sc-2025; Santa Cruz Biotechnology) for 2 hours at 4°C, followed by incubation with 30 μL protein G Seph agarose (10253638; GE Healthcare) overnight at 4°C.

Purification:

Article Title: WASP and Mst1 coregulate B-cell development and B-cell receptor signaling
Article Snippet: .. For coimmunoprecipitation, purified splenic B cells (5 × 106 ) were incubated with or without Biotin-conjugated F(ab')2 Anti-Mouse Ig (G+M) (115-066-068; Jackson ImmunoResearch Laboratories) for indicated times in the presence or absence of streptavidin. .. Cells were then lysed with a 150 μL radioimmunoprecipitation assay lysis buffer (Beyotime, P0013B) containing protease inhibitor cocktail (G2006; Servicebio).The cell lysates were incubated with 1 μg of antibody anti-Mst1 or control immunoglobulin G (sc-2025; Santa Cruz Biotechnology) for 2 hours at 4°C, followed by incubation with 30 μL protein G Seph agarose (10253638; GE Healthcare) overnight at 4°C.

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