Journal: Cell

Article Title: Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches

doi: 10.1016/j.cell.2021.12.018

Figure Lengend Snippet:

Article Snippet: Anti-Human CD177 (REA258) PE , Miltenyi Biotec , 130-101-525; RRID: AB_2655641.

Techniques: Purification, Recombinant, Staining, cDNA Synthesis, Gene Expression, Software, Microscopy

Journal: PLoS Genetics

Article Title: Heterogeneity of Human Neutrophil CD177 Expression Results from CD177P1 Pseudogene Conversion

doi: 10.1371/journal.pgen.1006067

Figure Lengend Snippet: A. Neutrophils of 5 subjects were gated on CD66b + cells and analysed for surface expression of CD177. Three neutrophil subsets defined by CD177 expression were shown: negative (neg) in black, intermediate (int) in orange and high (hi) in pink, percentages of each subset were indicated as numbers in the same colour. B . Atypical CD177 expression with CD177 int subset >20% of neutrophils. C. A dot plot showing percentages of CD177 hi versus CD177 neg of total neutrophils in healthy subjects, each dot represents one subject. Three major phenotypic groups were marked as CD177 hi , CD177 hi/neg , and CD177 null . D. Contour plots showing neutrophils from a CD177 null subject (left) and a tri-model CD177 expressing donor (right), co-stained with two CD177 specific monoclonal antibodies (MEM-166 and REA258).

Article Snippet: Leukocytes (approximately 1x10 6 ) were stained with fluorescent conjugated antibodies for CD16 (3G8), CD66b (G10F5) from Biolegend, CD177 (MEM-166) from Abcam and CD177 (REA258) from Miltenyl Biotec in Ca ++ Mg ++ free HBSS.

Techniques: Expressing, Staining, Bioprocessing

Journal: PLoS Genetics

Article Title: Heterogeneity of Human Neutrophil CD177 Expression Results from CD177P1 Pseudogene Conversion

doi: 10.1371/journal.pgen.1006067

Figure Lengend Snippet: A . Summary of deep sequencing of CD177 gene (located at 43.8Mb, band q13.2, of the forward strand of chromosome 19 in human genome 19, 43.3 Mb and q13.31 for hg38). Amplicons covering the entire coding sequence of 9 exons were sequenced. 100% coverage was obtained at indicated read depths. 24 low frequency SNPs were found in exon adjacent non-coding regions and 17 polymorphisms were identified in CD177 coding sequences. Representative allelic frequencies of nine SNPs in exon 5 and 7 are displayed according to CD177 phenotypes. Graphs were generated from Integrative Genomics Viewer from Broad Institute referenced on hg19. B . SNV frequency of 41 variants (called from hg19) in cohort 1. Each dashed line represents genotypes from a single individual. A schematic CD177 gene structure is shown of variants within CD177 . C . Sanger sequencing of CD177 exon 7 in DNA isolated from neutrophils (upper panel) and saliva (lower panel) from three individuals same as shown in A . Reference (Ref) and variant (Var) read frequencies derived from deep sequencing and CD177 neutrophil phenotypes for each subject were indicated on the top of each panel.

Article Snippet: Leukocytes (approximately 1x10 6 ) were stained with fluorescent conjugated antibodies for CD16 (3G8), CD66b (G10F5) from Biolegend, CD177 (MEM-166) from Abcam and CD177 (REA258) from Miltenyl Biotec in Ca ++ Mg ++ free HBSS.

Techniques: Sequencing, Generated, Isolation, Variant Assay, Derivative Assay

Journal: PLoS Genetics

Article Title: Heterogeneity of Human Neutrophil CD177 Expression Results from CD177P1 Pseudogene Conversion

doi: 10.1371/journal.pgen.1006067

Figure Lengend Snippet: List of 17 protein-coding variations showed their incidence, genomic location, polymorphisms and alteration in amino acids (A.A.) in GRCh 37 (hg19) and updated version GRCh38 (hg38).

Article Snippet: Leukocytes (approximately 1x10 6 ) were stained with fluorescent conjugated antibodies for CD16 (3G8), CD66b (G10F5) from Biolegend, CD177 (MEM-166) from Abcam and CD177 (REA258) from Miltenyl Biotec in Ca ++ Mg ++ free HBSS.

Techniques:

Journal: PLoS Genetics

Article Title: Heterogeneity of Human Neutrophil CD177 Expression Results from CD177P1 Pseudogene Conversion

doi: 10.1371/journal.pgen.1006067

Figure Lengend Snippet: A—C . Association of reference allele frequency (g.7497A) with neutrophil CD177 expression measured by geometric mean fluorescence intensity (MFI) of CD177 on the cell surface of total neutrophils ( A ), percentages of CD177 hi neutrophils ( B ), and CD177 neg neutrophils ( C ) in the blood of cohort 2. D . Heat map of variant allele frequency (determined by deep sequencing) and neutrophil phenotypes (determined by flow cytometry) in cohort 1.

Article Snippet: Leukocytes (approximately 1x10 6 ) were stained with fluorescent conjugated antibodies for CD16 (3G8), CD66b (G10F5) from Biolegend, CD177 (MEM-166) from Abcam and CD177 (REA258) from Miltenyl Biotec in Ca ++ Mg ++ free HBSS.

Techniques: Expressing, Fluorescence, Variant Assay, Sequencing, Flow Cytometry

Journal: PLoS Genetics

Article Title: Heterogeneity of Human Neutrophil CD177 Expression Results from CD177P1 Pseudogene Conversion

doi: 10.1371/journal.pgen.1006067

Figure Lengend Snippet: A . CD177 locus on human chromosome 19 and a schematic comparison of CD177 and CD177P1 genes. B. Current annotation of three copy number variations of CD177 and CD177P1 gene polymorphisms. C . CD177 reference allele frequencies of two polymorphisms, g.1991C in exon 4 and g.7497A in exon 7, of cohort 1. Each dot represents one of 40 tested subjects. 15 out of 40 subjects displayed allele frequencies of g.1991C and g.7497A simultaneously at 50%. 14/40 subjects harboured similarly 50% g.7497A allele but 75% g.1991C. D . Proposed CD177/CD177P1 haplotypes in two loci of exon 4 (C/G) and exon 7 (A/T). CD177 gene in black line and CD177P1 in grey. The most frequent genotype is highlighted.

Article Snippet: Leukocytes (approximately 1x10 6 ) were stained with fluorescent conjugated antibodies for CD16 (3G8), CD66b (G10F5) from Biolegend, CD177 (MEM-166) from Abcam and CD177 (REA258) from Miltenyl Biotec in Ca ++ Mg ++ free HBSS.

Techniques: Comparison

Journal: PLoS Genetics

Article Title: Heterogeneity of Human Neutrophil CD177 Expression Results from CD177P1 Pseudogene Conversion

doi: 10.1371/journal.pgen.1006067

Figure Lengend Snippet: A-D . Pedigrees of 4 unrelated families with different CD177 geno- and phenotypes, all exhibit Mendelian inheritance. ⊕: female, ⊠: male; filled: CD177 exon 7, blank: CD177P1 exon 7. Genotypes were also shown in schematic structures, CD177 in orange and CD177P1 in blue, with g.7497A/T polymorphisms labelled in both alleles at CD177 and CD177P1 loci. Chromatograms showed genomic sequence trace of CD177 g.7497 labelled with ratio of Ref:Var in each subject. Histograms showed CD177 phenotypes, numbers in corresponding colours indicating percentages of neutrophil subsets, CD177 neg in black, CD177 int in orange, CD177 hi in pink.

Article Snippet: Leukocytes (approximately 1x10 6 ) were stained with fluorescent conjugated antibodies for CD16 (3G8), CD66b (G10F5) from Biolegend, CD177 (MEM-166) from Abcam and CD177 (REA258) from Miltenyl Biotec in Ca ++ Mg ++ free HBSS.

Techniques: Sequencing

Journal: PLoS Genetics

Article Title: Heterogeneity of Human Neutrophil CD177 Expression Results from CD177P1 Pseudogene Conversion

doi: 10.1371/journal.pgen.1006067

Figure Lengend Snippet: A . CD177 neg and CD177 hi cells were sorted from an individual who predominantly express CD177 hi in neutrophils ( left ). Sequencing traces showed CD177 codon variations at indicated loci, comparing results from gDNA deep sequencing (top), CD177 mRNA isolated from purified CD177 hi cells (middle) and CD177 neg neutrophils (bottom). The reference nucleotide sequences were labelled in colour letters below. The two SNPs present in gDNA and cDNA of both cell subsets were labelled (*). B . A schematic summary of the variations in gDNA and mRNA in neutrophils of this subject. Sequence variations were indicated on the top of the gene/transcript and exons were indicated (E2, E4 etc) below. CD177P1 nucleotides in exon 4, 5 and 7 were outlined. CD177P1 transcripts were shown in grey signifying expected NMD with the stop codon variation g.7497T in red.

Article Snippet: Leukocytes (approximately 1x10 6 ) were stained with fluorescent conjugated antibodies for CD16 (3G8), CD66b (G10F5) from Biolegend, CD177 (MEM-166) from Abcam and CD177 (REA258) from Miltenyl Biotec in Ca ++ Mg ++ free HBSS.

Techniques: Sequencing, Isolation, Purification

Journal: PLoS Genetics

Article Title: Heterogeneity of Human Neutrophil CD177 Expression Results from CD177P1 Pseudogene Conversion

doi: 10.1371/journal.pgen.1006067

Figure Lengend Snippet: A . CD177 hi and CD177 neg neutrophils were sorted from a single donor with bimodal CD177 expression ( left ). Genomic variant allele frequencies were determined by deep sequencing ( top ), and compared with sequence variations in cDNA from CD177 hi ( middle ) and CD177 neg neutrophils ( bottom ). Two CD177 transcripts were found in CD177 neg neutrophils. SNPs present in gDNA and cDNA of CD177 neg subsets but absent from CD177 hi cells are labelled (*). B . Schematic summaries of CD177 vs CD177P1 gDNA variations and CD177 mRNA in two neutrophil subsets. CD177P1 derived nucleotides in exon 4, 5 and 7 are outlined, suggesting one CD177 allele partially supplied by CD177P1 exon 7. C & E . Genomic sequence traces in indicated loci within exon 4, 5 and 7 of two CD177 null individuals. Only CD177P1 exon 7 sequences are detected in CD177 null subjects, who harbour both CD177 and CD177P1 upstream elements, i.e., exon 4 ( C ) and exon 5 ( E ). D & F . Schematic genomic CD177 / CD177P1 structures of CD177 null individuals as shown in C & E . G . Confirmation of ectopic and allelic CD177P1 exon 7 conversion in three subjects by MLPA. The plots show the peak ratio of probes for indicated loci of CD177 and CD177P1 genes. Exon 2 is used as a reference read out of the CD177 gene only; probes for exon 4, 5, 7 and 9 bind to both CD177 and CD177P1 , whereas probes labelled as (P1) are specific to CD177P1 exon 5 and 7. The graph shows one copy duplication of CD177P1 exon 7 in blue in the same subject as shown in A & B ; and two copies duplication (allelic conversion) in the two CD177 null subjects (red & orange) as shown in C-F . The subject in red also shows duplication of CD177P1 exon 5 in concordant to C & D .

Article Snippet: Leukocytes (approximately 1x10 6 ) were stained with fluorescent conjugated antibodies for CD16 (3G8), CD66b (G10F5) from Biolegend, CD177 (MEM-166) from Abcam and CD177 (REA258) from Miltenyl Biotec in Ca ++ Mg ++ free HBSS.

Techniques: Expressing, Variant Assay, Sequencing, Derivative Assay

Journal: PLoS Genetics

Article Title: Heterogeneity of Human Neutrophil CD177 Expression Results from CD177P1 Pseudogene Conversion

doi: 10.1371/journal.pgen.1006067

Figure Lengend Snippet: A . A proposed allelic arrangement model to account for CD177 variant read frequencies. CD177P1 -derived segments are shown in blue boxes and g.7497T in red lines. CD177 genes are in yellow and g.7497A in green. Prevalence of each arrangement in cohort 2 is shown, together with calculated Hardy-Weinberg ratio. B . Model to account for observed CD177 genotype-phenotype relation.

Article Snippet: Leukocytes (approximately 1x10 6 ) were stained with fluorescent conjugated antibodies for CD16 (3G8), CD66b (G10F5) from Biolegend, CD177 (MEM-166) from Abcam and CD177 (REA258) from Miltenyl Biotec in Ca ++ Mg ++ free HBSS.

Techniques: Variant Assay, Derivative Assay