monoclonal gr1  (Absolute Biotech Inc)

Journal: Frontiers in Medicine

Article Title: Blockade of IL-18Rα-mediated signaling pathway exacerbates neutrophil infiltration in imiquimod-induced psoriasis murine model

doi: 10.3389/fmed.2023.1293132

Figure Lengend Snippet: Il18ra -mediated signals may suppress IL-17 production by neutrophils activation. Infiltration of Gr1 positive cells and citrullinated histon 3 (NETs) into skin tissues of Il18ra −/− applied IMQ (A,B) . Photomicrographs were taken at 10× magnification to compare the area of Gr1 positive cells and NETs infiltration into skin lesions between Il18ra −/− and WT mice treated with IMQ. Increased infiltration of Gr1 positive cells and NETs in Il18ra −/− were observed ( C,D : n = 7). Gr1 positive cell infiltration and serum IL-17A levels correlate moderate and NETs and Il17a mRNA levels in skin lesion also have correlation (Gr1, R 2 = 0.4480, p = 0.0173; NETs, R 2 = 0.6828, p = 0.0009) ( E,F : n = 12). The data are expressed as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. IL-17A, interuikin-17A; l18ra, interleukin-18 receptor alpha; NETs, neutrophil extracellular traps; WT, wild type; IMQ, imiquimod.

Article Snippet: Slides were incubated overnight at 4°C with the following primary antibodies: monoclonal antibody for Gr1/Ly6C (1:100) (Pharmingen, San Diego, CA, United States), rat monoclonal antibody GK1.5 for CD4 (1:100) (Pharmingen), F4/80 hybridoma culture supernatant (1:2.5) (HB 198; American Type Culture Collection, Manassas, MD, United States), mouse monoclonal antibody for CD11c (1:100) (Abcam, Cambridge, United Kingdom), and rabbit polyclonal antibody for citrullinated histone H3 (1:200) (Abcam, Cambridge, United Kingdom).

Techniques: Activation Assay

Journal: eLife

Article Title: Haploinsufficiency of the essential gene Rps12 causes defects in erythropoiesis and hematopoietic stem cell maintenance

doi: 10.7554/eLife.69322

Figure Lengend Snippet:

Article Snippet: Antibody , APC-Cy7 Rat anti-mouse monoclonal Gr1 , BD biosciences , Cat # 557661 , Clone: RB6-8C5 (1 µl per 10 7 cells).

Techniques: Generated, Staining, Sequencing, DNA Extraction, Western Blot, Isolation, SYBR Green Assay

Journal: eLife

Article Title: A lncRNA identifies Irf8 enhancer element in negative feedback control of dendritic cell differentiation

doi: 10.7554/eLife.83342

Figure Lengend Snippet: ( A ) Genomic anatomy of lncIrf8 locus determined by 3’ and 5’ RACE PCR. Blue box, exon 2 and 3 (48 bp and 468 bp, respectively). The 1010 bp intron and polyA tail are shown. Data of RNA-seq, ATAC-seq, ChIP-seq of H3K27ac (enhancer mark) and H3K4me3 (active promoter mark, near TSS) are visualized by IGV browser for the indicated cell populations (pDC, all cDC, cDC1 and cDC2). Grey box, lncIrf8 promoter KO region; open box, cDC1 specific +32 kb enhancer by . Irf8 +32 kb enhancer based on the H3K27ac enhancer mark is indicated with a purple line. Scale bar: 1 kb. ( B ) Gene expression of lncIrf8 and Irf8 in lncIrf8 promoter KO and control at day 0, 5, and 7 of Flt3L directed DC differentiation. Gene expression was determined by RT-qPCR and normalized to GAPDH . n=4. ( C ) Representative flow cytometry analysis of Flt3L directed DC differentiation of lncIrf8 promoter KO HoxB8 MPP and control ( ; ). pDC, all cDC, cDC1, and cDC2 were gated as in and are shown. Bar diagrams depict quantification of pDC, cDC1 and cDC2 normalized to living single cells on DC differentiation day 0, 3, 5, 7, and 9. n=6–7. ( D ) Representative phase-contrast microscopy images of lncIrf8 promoter KO HoxB8 MPP and control on day 7 of Flt3L directed DC differentiation. Scale bar: 200 μm. ( E ) Representative flow cytometry analysis of spontaneous DC differentiation of lncIrf8 promoter KO HoxB8 MPP and control with growth factors but without E2 ( ; ) at day 8. Gr1 + monocytes and CD11c + DC are shown. Quantification of Gr1 + monocytes of living single cells on day 3, 6, 8, and 10 of spontaneous DC differentiation. n=6, lncIrf8 promoter KO; n=4, control. Empty gRNA vector or non-targeting gRNA vector HoxB8 MPP were used as controls. Data represent mean ± SD of at least three independent experiments with different HoxB8 MPP clones of lncIrf8 promoter KO and control without deletion. *p<0.05, **p<0.01, ***p<0.001, multiple t-tests. Data that have no difference (p>0.05) are not labeled.

Article Snippet: Antibody , PerCP/Cyanine 5.5 anti-mouse Gr1 (rat monoclonal) , eBioscience , Cat# 45-5931-80; RRID: AB_906247 , FACS (1:400).

Techniques: RNA Sequencing, ChIP-sequencing, Gene Expression, Control, Quantitative RT-PCR, Flow Cytometry, Microscopy, Plasmid Preparation, Clone Assay, Labeling

Journal: eLife

Article Title: A lncRNA identifies Irf8 enhancer element in negative feedback control of dendritic cell differentiation

doi: 10.7554/eLife.83342

Figure Lengend Snippet: ( A ) Gating strategy for spontaneous DC differentiation. Gr1 + monocytes, and the pDC, all cDC, cDC1 and cDC2 subsets from Gr1 - CD11c + cells were gated as in . ( B ) Representative phase-contrast microscopy images of lncIrf8 promoter KO HoxB8 MPP and control on spontaneous DC differentiation (-E2) day 8. Scale bar: 200 μm. ( C–G ) Quantification of CD11c + DC, pDC, all cDC, cDC1 and cDC2 of panel ( A ) of living single cells in lncIrf8 promoter KO HoxB8 MPP and control on spontaneous DC differentiation (-E2) day 3, 6, 8, and 10. n=6 for lncIrf8 promoter KO; n=4 for KO control. Data represent mean ± SD of at least three independent experiments with different HoxB8 MPP clones of lncIrf8 promoter KO and control without deletion. *p<0.05, **p<0.01, ***p<0.001, multiple t-tests. Data that have no difference (p>0.05) are not labeled. ( H ) Quantification of cDC2 normalized to CD11c + cells of ( G ) at days 6, 8, and 10. Spontaneous DC differentiation of control cells progressed slower than differentiation of lncIrf8 promoter KO cells and therefore a lower frequency of cDC2 was observed at day 6. At later time points lncIrf8 promoter KO left cDC2 differentiation unaffected. ( I ) Gating strategy for identification of Gr1 + monocytes upon spontaneous DC differentiation in panel ( A ) and . Mouse BM cells were used and Gr1 + monocytes (Mo) were gated as 7-AAD - CD11b + CD11c - Ly6C + Ly6G - . Neutrophils (Neut) were gated as 7-AAD - CD11b + CD11c - Ly6C low/- Ly6G + . ( J ) Representative flow cytometry analysis of Gr1 + monocytes at day 6 of spontaneous DC differentiation of lncIrf8 promoter KO and control. Cells were gated as in panel ( I ).

Article Snippet: Antibody , PerCP/Cyanine 5.5 anti-mouse Gr1 (rat monoclonal) , eBioscience , Cat# 45-5931-80; RRID: AB_906247 , FACS (1:400).

Techniques: Microscopy, Control, Clone Assay, Labeling, Flow Cytometry

Journal: eLife

Article Title: A lncRNA identifies Irf8 enhancer element in negative feedback control of dendritic cell differentiation

doi: 10.7554/eLife.83342

Figure Lengend Snippet: ( A ) Position of cDC1 specific +32 kb enhancer. Data of ATAC-seq, ChIP-seq of H3K27ac (enhancer mark) are visualized by IGV browser for the indicated cell populations (pDC, all cDC, cDC1, and cDC2). Open box, cDC1 specific +32 kb enhancer by ; grey box, lncIrf8 promoter KO region; Irf8 +32 kb enhancer based on the H3K27ac enhancer mark is indicated with a purple line. Scale bar: 1 kb. ( B and C ) Representative genotyping of cDC1 specific +32 kb enhancer KO in single-cell Mx-Cas9-GFP HoxB8 MPP. Homozygous deletions in cDC1 specific +32 kb enhancer KO (red) were detected by PCR and agarose gel electrophoresis ( B ). cDC1 specific +32 kb enhancer KO deletions determined by Sanger sequencing in five representative KO clones of ( B ) are depicted in ( C ). cDC1 +32 kb-F1 and cDC1 +32 kb-R refers to primers for genotyping. Sanger sequencing was performed from on both DNA strands: cDC1 +32 kb-F2 and cDC1 +32 kb-R was used for forward and reverse sequencing, respectively. Clones 1–14, 1–35, 1–51, 1–61, and 2–56 are shown. Positions of gRNAs used for generating the deletion of cDC1 +32 kb enhancer KO are indicated. gRNA-KO1-5' and gRNA-KO1-3' are from ; gRNA-KO2-5' and gRNA-KO2-3' were designed in the present study. ( D ) Gene expression of lncIrf8 and Irf8 in cDC1 specific +32 kb enhancer KO and control at day 0 and 7 of Flt3L directed DC differentiation. n=6, cDC1 specific +32 kb enhancer KO; n=4, control. ( E ) Representative flow cytometry analysis of Flt3L directed DC differentiation of cDC1 specific +32 kb enhancer KO HoxB8 MPP and control. pDC, all cDC, cDC1, and cDC2 were gated as in and are shown. Bar diagrams on the left depict quantification of pDC, cDC1 and cDC2 normalized to CD11c + cells on DC differentiation day 7 and 9. Bar diagrams on the right depict CD11c + DC and all cDC. CD11c + DC were gated on living single cells. Frequencies of pDC were compromised in +32 kb enhancer KO at day 7, however this was not statistically significant. n=6, cDC1 specific +32 kb enhancer KO; n=4, control. ( F ) Representative flow cytometry analysis of spontaneous DC differentiation of cDC1 specific +32 kb enhancer KO and control with growth factors but without E2 at day 8. Gr1 + monocytes and CD11c + DC are shown. Quantification of Gr1 + monocytes of living single cells on days 3, 6, 8, and 10 of spontaneous DC differentiation. n=6, cDC1 specific +32 kb enhancer KO; n=4, control. Empty gRNA vector or non-targeting gRNA vector HoxB8 MPP were used as controls. Data represent mean ± SD of three independent experiments with different HoxB8 MPP clones of cDC1 specific +32 kb enhancer KO and control without deletion. Differentiation kinetics of clones were normatized to control without deletion if required. *p<0.05, **p<0.01, ****p<0.0001, multiple t-tests. Data that have no difference (p>0.05) are not labeled. Figure 2—figure supplement 4—source data 1. Full raw unedited gel of genotyping of cDC1 specific +32 kb enhancer KO clones in . Figure 2—figure supplement 4—source data 2. Labeled uncropped gel of genotyping of cDC1 specific +32 kb enhancer KO clones in .

Article Snippet: Antibody , PerCP/Cyanine 5.5 anti-mouse Gr1 (rat monoclonal) , eBioscience , Cat# 45-5931-80; RRID: AB_906247 , FACS (1:400).

Techniques: ChIP-sequencing, Agarose Gel Electrophoresis, Sequencing, Clone Assay, Gene Expression, Control, Flow Cytometry, Plasmid Preparation, Labeling

Journal: eLife

Article Title: A lncRNA identifies Irf8 enhancer element in negative feedback control of dendritic cell differentiation

doi: 10.7554/eLife.83342

Figure Lengend Snippet: ( A ) Workflow of HoxB8 MPP transplantation. CD45.1 recipient mice were sublethal irradiated and injected with CD45.2 lncIrf8 promoter KO and control HoxB8 MPP. Seven and 14 days after cell transplantation, mice were sacrificed and cells from bone marrow and spleen were isolated and subjected to flow cytometry analysis for DC subsets. ( B ) Gating strategy for CD45.2 DC subsets following transplantation of CD45.2 lncIrf8 promoter KO HoxB8 MPP into CD45.1 recipient mice. 7-AAD - CD45.2 + lineage - donor cells were gated on Gr1 + monocytes, Gr1 - Siglec H + pDC, and Gr1 - Siglec H - CD11c + MHCII + DC, and further on cDC1 and cDC2 subsets. cDC1 are Gr1 - Siglec H - MHCII + CD11c + CD11b low/- XCR1 + and cDC2 are Gr1 - Siglec H - MHCII + CD11c + CD11b + XCR1 - . The lineage cocktail (Lin) was: Ter119, CD19, CD3e, NK1.1, F4/80.

Article Snippet: Antibody , PerCP/Cyanine 5.5 anti-mouse Gr1 (rat monoclonal) , eBioscience , Cat# 45-5931-80; RRID: AB_906247 , FACS (1:400).

Techniques: Transplantation Assay, Irradiation, Injection, Control, Isolation, Flow Cytometry

Journal: eLife

Article Title: A lncRNA identifies Irf8 enhancer element in negative feedback control of dendritic cell differentiation

doi: 10.7554/eLife.83342

Figure Lengend Snippet: ( A ) Representative flow cytometry analysis of CD45.2 lncIrf8 promoter KO and control HoxB8 MPP in BM at day 7 after cell transplantation (for details see ). Donor cell populations were gated from 7-AAD - CD45.2 + Lin - cells and Gr1 + monocytes, pDC, cDC1 and cDC2 are shown. ( B–F ) Quantification of Gr1 + monocytes, MHCII + CD11c + DC, pDC, cDC1, and cDC2 of living single cells in BM on day 7 and 14 after cell transplantation (n=3–4). ( G ) Representative flow cytometry analysis of lncIrf8 promoter KO and control HoxB8 MPP in spleen at day 7 after cell transplantation. Gating was as in panel ( A ). ( H–L ) Quantification of Gr1 + monocytes, MHCII + CD11c + DC, pDC, cDC1 and cDC2 on day 7 and 14 after cell transplantation (n=3–4). Data represent mean ± SD from 3 to 4 mice. *p<0.05, **p<0.01, ***p<0.001, multiple t-tests. Data that have no difference (p>0.05) are not labeled.

Article Snippet: Antibody , PerCP/Cyanine 5.5 anti-mouse Gr1 (rat monoclonal) , eBioscience , Cat# 45-5931-80; RRID: AB_906247 , FACS (1:400).

Techniques: Flow Cytometry, Control, Transplantation Assay, Labeling

Journal: eLife

Article Title: A lncRNA identifies Irf8 enhancer element in negative feedback control of dendritic cell differentiation

doi: 10.7554/eLife.83342

Figure Lengend Snippet:

Article Snippet: Antibody , PerCP/Cyanine 5.5 anti-mouse Gr1 (rat monoclonal) , eBioscience , Cat# 45-5931-80; RRID: AB_906247 , FACS (1:400).

Techniques: Control, Over Expression, Activation Assay, Activity Assay, Recombinant, Saline, Expressing, Fluorescence, Reverse Transcription, Multiplex Assay, TA Cloning, Sequencing, Cloning, Capture-C, Software

Journal: Cancers

Article Title: Phototheranostics of Splenic Myeloid-Derived Suppressor Cells and Its Impact on Spleen Metabolism in Tumor-Bearing Mice

doi: 10.3390/cancers14153578

Figure Lengend Snippet: Accumulation of splenic MDSCs in tumor-bearing mice. ( a ) Representative flow cytometric analysis comparing CD11b and Gr1 expression in splenocytes from non-tumor-bearing (NTB) to 4T1-luc tumor-bearing (TB) four- to six-week-old female BALB/c mice at 3–4 weeks post-tumor implantation. ( b ) Percentage of CD11b + Gr1 + MDSCs in the spleen of NTB ( n = 3) and TB ( n = 4) mice. Values represent mean ± SEM. Two-tailed Student’s t -test. *** p ≤ 0.001.

Article Snippet: Anti-Gr1 monoclonal antibody (clone: RB6-8C5) and monoclonal rat IgG2b,κ antibody (clone: LTF-2) were obtained from BioXCell (Lebanon, NH, USA).

Techniques: Expressing, Tumor Implantation, Two Tailed Test

Journal: Cancers

Article Title: Phototheranostics of Splenic Myeloid-Derived Suppressor Cells and Its Impact on Spleen Metabolism in Tumor-Bearing Mice

doi: 10.3390/cancers14153578

Figure Lengend Snippet: Gr1-IR700 eliminates tumor-induced splenic MDSCs in vitro. ( a ) Schematic of Gr1-IR700 synthesis. ( b ) Representative flow cytometric analysis of freshly isolated and MACS-enriched (84.5% ± 2.4%) Gr1 + splenocytes ( n = 4). ( c ) Gr1-IR700-mediated PIT reduced cell viability at a fixed NIR light dose of 16 J/cm 2 and increasing concentration of Gr1-IR700 ( n = 4). ( d ) Gr1-IR700-mediated cell killing increased in a NIR-light dose-dependent manner ( n = 4). ( e ) Selective Gr1-IR700-PIT-mediated cell killing compared to NIR-irradiated PBS and IgG-IR700 groups. Phototoxicity was partially inhibited after 5-fold excess pre-incubation using unconjugated anti-Gr1. Values represent mean ± SEM. Each of the figure panels represents at least 3 experimental replicates.

Article Snippet: Anti-Gr1 monoclonal antibody (clone: RB6-8C5) and monoclonal rat IgG2b,κ antibody (clone: LTF-2) were obtained from BioXCell (Lebanon, NH, USA).

Techniques: In Vitro, Isolation, Concentration Assay, Irradiation, Incubation

Journal: Cancers

Article Title: Phototheranostics of Splenic Myeloid-Derived Suppressor Cells and Its Impact on Spleen Metabolism in Tumor-Bearing Mice

doi: 10.3390/cancers14153578

Figure Lengend Snippet: Gr1-IR700 preferentially accumulates in the spleens and lungs of TB mice. ( a ) Representative in vivo fluorescence images obtained before and after administration of Gr1-IR700 in NTB mouse (top), IgG-IR700 in TB mouse (middle), and Gr1-IR700 in TB mouse (bottom) and corresponding ex vivo fluorescence images of the small intestine (SI), liver (Li), lung (Lu), stomach (St), blood (B), spleen (Sp), kidney (K) and heart (H). Also included is ex vivo fluorescence from tumor slices at 1 mm thickness (right). ( b ) Biodistribution from Mean Fluorescence Intensity (MFI) of Gr1-IR700 and IgG-IR700 in Gr1-IR700-injected NTB mice ( n = 4), IgG-IR700-injected TB mice ( n = 4) and Gr1-IR700-injected TB mice ( n = 3) at 24 h post-intraperitoneal injection. MFI values indicate significantly higher MFI in the spleen, lungs and blood of TB mice injected with Gr1-IR700. Two-tailed Student’s t -test. * p ≤ 0.05, # p ≤ 0.01, and + p ≤ 0.001. ( c ) Hematoxylin and eosin stained 5 µm thick lung sections from three Gr1-IR700-injected TB mice at 4X (left), 10X (middle) and 40X (right) magnification, with the corresponding MFI in the lung sections displayed adjacent to the 40X section. MFI was highest in the lungs with the highest number of nodules.

Article Snippet: Anti-Gr1 monoclonal antibody (clone: RB6-8C5) and monoclonal rat IgG2b,κ antibody (clone: LTF-2) were obtained from BioXCell (Lebanon, NH, USA).

Techniques: In Vivo, Fluorescence, Ex Vivo, Injection, Two Tailed Test, Staining

Journal: Cancers

Article Title: Phototheranostics of Splenic Myeloid-Derived Suppressor Cells and Its Impact on Spleen Metabolism in Tumor-Bearing Mice

doi: 10.3390/cancers14153578

Figure Lengend Snippet: In vivo Gr1-IR700-mediated PIT of tumor-bearing mouse spleens. ( a ) Representative in vivo fluorescence images of mice pre-injection and at 24 h and 48 h post-IP injection of Gr1-IR700 without light (top) or exposed to NIR light (bottom). NIR-PIT was administered at an exposure dose of 420 J/cm 2 with a red light-emitting diode (LED) at a peak wavelength of 690 nm. Representative images from mice that were further imaged at 4 h post-PIT are also shown. Dark and PIT correspondingly refer to non-irradiated TB mice and NIR-irradiated TB mice. Exposure to light resulted in a decrease in fluorescence due to the breakdown of IR700. ( b ) Representative ex vivo fluorescence image of excised mouse spleen from the Dark (left) and PIT group (right) confirmed the loss of IR700-fluorescence in the PIT-treated spleen due to NIR light exposure. ( c ) Cell viability of splenocytes measured using 5 × 10 5 splenocytes from excised spleens of the uninjected TB Ctrl ( n = 3), Dark ( n = 4) and PIT ( n = 7) groups incubated in CCK-8 solution for 90–100 min. Two-tailed Student’s t -test. *** p ≤ 0.001.

Article Snippet: Anti-Gr1 monoclonal antibody (clone: RB6-8C5) and monoclonal rat IgG2b,κ antibody (clone: LTF-2) were obtained from BioXCell (Lebanon, NH, USA).

Techniques: In Vivo, Fluorescence, Injection, Irradiation, Ex Vivo, Incubation, CCK-8 Assay, Two Tailed Test

Journal: Cancers

Article Title: Phototheranostics of Splenic Myeloid-Derived Suppressor Cells and Its Impact on Spleen Metabolism in Tumor-Bearing Mice

doi: 10.3390/cancers14153578

Figure Lengend Snippet: Representative 1 H-MR spectra of aqueous spleen metabolites from NTB (black), tumor-bearing Ctrl (green), Gr1-IR700-injected Dark (blue) and Gr1-IR700-injected PIT (red) mice obtained 4 h post-PIT. BHB—beta-hydroxybutyrate; PC—phosphocholine; GPC—glycerophosphocholine.

Article Snippet: Anti-Gr1 monoclonal antibody (clone: RB6-8C5) and monoclonal rat IgG2b,κ antibody (clone: LTF-2) were obtained from BioXCell (Lebanon, NH, USA).

Techniques: Injection

Journal: Cancers

Article Title: Phototheranostics of Splenic Myeloid-Derived Suppressor Cells and Its Impact on Spleen Metabolism in Tumor-Bearing Mice

doi: 10.3390/cancers14153578

Figure Lengend Snippet: Heat map of aqueous spleen metabolites from NTB ( n = 5), TB Ctrl ( n = 5), Gr1-IR700-injected Dark ( n = 5) and Gr1-IR700-injected PIT ( n = 5) mice 4 h post-PIT. Bold font indicates significant percent changes with two-tailed Student’s t -test, p ≤ 0.05. * denotes NTB vs. Ctrl, + denotes Ctrl vs. Dark, # denotes Ctrl vs. PIT, x denotes Dark vs. PIT and ^ denotes NTB vs. PIT. BHB—beta-hydroxybutyrate; PC—phosphocholine; GPC—glycerophosphocholine. The red boxes denote metabolites in the spleen of TB mice that normalized to NTB levels following PIT.

Article Snippet: Anti-Gr1 monoclonal antibody (clone: RB6-8C5) and monoclonal rat IgG2b,κ antibody (clone: LTF-2) were obtained from BioXCell (Lebanon, NH, USA).

Techniques: Injection, Two Tailed Test

Journal: Cancers

Article Title: Phototheranostics of Splenic Myeloid-Derived Suppressor Cells and Its Impact on Spleen Metabolism in Tumor-Bearing Mice

doi: 10.3390/cancers14153578

Figure Lengend Snippet: 1 H MRS analysis of spleen metabolite concentrations from non-tumor-bearing NTB ( n = 5), TB Ctrl ( n = 5), Gr1-IR700-injected Dark ( n = 5) and Gr1-IR700-injected PIT ( n = 5) mice 4 h post-PIT. Bold font indicates significant percent changes evaluated with two-tailed Student’s t -test comparing Ctrl vs. PIT, Dark vs. PIT, NTB vs. Ctrl and Ctrl vs. Dark. Values represent mean ± SEM, n = 5 per group. BHB—beta-hydroxybutyrate; PC—phosphocholine; GPC—glycerophosphocholine.

Article Snippet: Anti-Gr1 monoclonal antibody (clone: RB6-8C5) and monoclonal rat IgG2b,κ antibody (clone: LTF-2) were obtained from BioXCell (Lebanon, NH, USA).

Techniques: Concentration Assay