Journal: Current protocols in immunology

Article Title: Protocols for the analysis of microRNA expression, biogenesis and function in immune cells

doi: 10.1002/cpim.78

Figure Lengend Snippet: Measurement of the expression of hsa-miR-15b-5p by reverse transcription and quantitative real time PCR (RT-qPCR). a) Reverse transcribed cDNAs are used for a quantitative real time PCR analysis (qPCR). An amplification chart of qPCR for hsa-miR-15b-5p (blue) and U6 (internal control, pink). b) RNAs isolated from RNA immunoprecipitation (RIP) complexes by an anti-pan Ago monoclonal antibody and an isotype control were reverse transcribed into cDNA. The enrichment of hsa-miR-15b-5p from both were detected by qPCR. A table of threshold cycles (Ct) for the amplification of hsa-miR-15b-5p under both conditions. ΔCT=CtAgo-CtIso. c) Relative fold enrichment of hsa-miR-15b-5p in isotype IgG RIP vs. anti-pan-Ago RIP. The enrichment of hsa-miR-15b-5p significantly increased in the anti-pan-Ago RIP complex.

Article Snippet: Materials Primers for mature miRNA or pre-miRNA or pri-miRNA - Human MicroRNA Assay kit (Qiagen) miScript II RT kit (Qiagen, Cat.# 218161 list-behavior=simple prefix-word= mark-type=none max-label-size=0 miScript Reverse Transcriptase Mix (optimized blend of poly(A) polymerase and reverse transcriptase) 10x miScript nucleics Mix 5x miScript HiSpec buffer 5x miScript HiFlex Buffer miScript SYBR Green PCR kit (Qiagen, Cat.# 218073) 2x QuantiTect SYBR Green PCR Master Mix 10x miScript universal primer (included in the kit) Hs_miR-15b miScript Primer assay (Qiagen, Cat.# MS00003185) Hs_RNU6 miScript Primer Assay (Qiagen, Cat.# MS00033740) Tabletop centrifuge with swinging bucket A Real Time PCR instrument (Bio-Rad, IQ5) Unskirted 96-Well PCR Plates (Bio-Rad, MLL9601) Microseal ‘B’ PCR Plate Sealing Film, adhesive, optical (Bio-Rad, msb1001) RNase free water Reverse transcription of total RNA to generate cDNA list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 Conduct basic protocol 1 steps 1–18 on 10 6 human B cells.

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Amplification, Control, Isolation, RNA Immunoprecipitation

Journal: Biomedicines

Article Title: Soluble JAM-C Ectodomain Serves as the Niche for Adipose-Derived Stromal/Stem Cells

doi: 10.3390/biomedicines9030278

Figure Lengend Snippet: JAM-B and JAM-C are concentrated on cell membranes of mouse adipose-derived stromal/stem cells (ADSCs). RT-PCR ( A ) and Western blot ( B ) for the indicated molecules in ADSCs derived from three mice (#1–3). Mouse kidney (for Jam-4 ) and spleen tissues (for other junctional adhesion molecules; JAMs) are used as positive controls (Ctrl). P, passage. ( C ) Confocal images of ADSCs stained for the indicated markers. Arrowheads show JAM-B- and JAM-C-immunoreactive signals on the cell membranes of ADSCs. Squares indicate the enlarged areas. Scale bar, 50 µm.

Article Snippet: To prepare the recombinant protein-coated plates, 1 µg of recombinant JAM-B (rJAM-B; 50464-M08H, SinoBiological, Beijing, China), recombinant JAM-C (rJAM-C; 50465-M08H, SinoBiological, Beijing, China), normal mouse IgG (12-371, Merck Millipore, Burlington, MA, USA) or Cellmatrix type I-A collagen (KP-2020, Nitta gelatin, Osaka, Japan) was placed in 12-well plates overnight at 4 °C.

Techniques: Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot, Staining

Journal: Biomedicines

Article Title: Soluble JAM-C Ectodomain Serves as the Niche for Adipose-Derived Stromal/Stem Cells

doi: 10.3390/biomedicines9030278

Figure Lengend Snippet: Fluorescence-activated cell sorting (FACS) profiles of the stromal vascular fraction (SVF) of the mouse adipose tissue. ( A ) Association between the expression of mesenchymal stem cell (MSC) markers CD44, CD105, CD140a and Sca-1 and JAM-B or JAM-C expression. ( B ) The JAM-C expression in the Sca1 /CD31 − /CD45 − /Ter119 − cell lineage.

Article Snippet: To prepare the recombinant protein-coated plates, 1 µg of recombinant JAM-B (rJAM-B; 50464-M08H, SinoBiological, Beijing, China), recombinant JAM-C (rJAM-C; 50465-M08H, SinoBiological, Beijing, China), normal mouse IgG (12-371, Merck Millipore, Burlington, MA, USA) or Cellmatrix type I-A collagen (KP-2020, Nitta gelatin, Osaka, Japan) was placed in 12-well plates overnight at 4 °C.

Techniques: Fluorescence, FACS, Expressing

Journal: Biomedicines

Article Title: Soluble JAM-C Ectodomain Serves as the Niche for Adipose-Derived Stromal/Stem Cells

doi: 10.3390/biomedicines9030278

Figure Lengend Snippet: Soluble JAM-C functions as the niche for ADSCs. ( A ) Schematic illustration of recombinant JAM-B (rJAM-B) and rJAM-C. S, signal domain; EC, extracellular domain; TM, transmembrane domain; C, cytoplasmic domain. ( B ) Cell adhesion assay for ADSCs grown on culture dishes coated with the indicated proteins. The relative levels are shown in histograms (mean ± SD; n = 5). ( C ) BrdU assay for ADSCs grown on culture plates coated with the indicated proteins. The BrdU/DAPI levels are shown in histograms (mean ± SD; n = 12). Scale bar, 100 µm. ( D ) RT-qPCR for the indicated MSC markers in ADSCs grown on culture dishes coated with the indicated proteins. The relative expression levels are shown in the histograms (mean ± SD; n = 4). * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: To prepare the recombinant protein-coated plates, 1 µg of recombinant JAM-B (rJAM-B; 50464-M08H, SinoBiological, Beijing, China), recombinant JAM-C (rJAM-C; 50465-M08H, SinoBiological, Beijing, China), normal mouse IgG (12-371, Merck Millipore, Burlington, MA, USA) or Cellmatrix type I-A collagen (KP-2020, Nitta gelatin, Osaka, Japan) was placed in 12-well plates overnight at 4 °C.

Techniques: Recombinant, Cell Adhesion Assay, BrdU Staining, Quantitative RT-PCR, Expressing

Journal: Biomedicines

Article Title: Soluble JAM-C Ectodomain Serves as the Niche for Adipose-Derived Stromal/Stem Cells

doi: 10.3390/biomedicines9030278

Figure Lengend Snippet: Association between soluble JAM-C and full-length JAM-B on ADSCs. Whole-cell lysates (1% of the input protein samples) and the samples immunoprecipitated (IP) with IgG or JAM-B Ab were immunoblotted (IB) with the indicated Abs. N.S., nonspecific signals.

Article Snippet: To prepare the recombinant protein-coated plates, 1 µg of recombinant JAM-B (rJAM-B; 50464-M08H, SinoBiological, Beijing, China), recombinant JAM-C (rJAM-C; 50465-M08H, SinoBiological, Beijing, China), normal mouse IgG (12-371, Merck Millipore, Burlington, MA, USA) or Cellmatrix type I-A collagen (KP-2020, Nitta gelatin, Osaka, Japan) was placed in 12-well plates overnight at 4 °C.

Techniques: Immunoprecipitation

Journal: Biomedicines

Article Title: Soluble JAM-C Ectodomain Serves as the Niche for Adipose-Derived Stromal/Stem Cells

doi: 10.3390/biomedicines9030278

Figure Lengend Snippet: A Soluble JAM-C couples with JAM-B to promote the ADSC adhesion and niche function. ( A ) Knockdown of the Jam2 gene encoding mouse JAM-B in ADSCs using the CRISPR method. ( B ) Western blot for the indicated proteins in the whole-cell lysates of the revealed ADSCs. ( C ) Cell adhesion assay for ADSCs grown in the indicated culture conditions. The relative levels are shown in histograms (mean ± SD; n = 8). ( D ) RT-qPCR for the indicated MSC markers in ADSCs cultivated in the indicated culture conditions. The relative expression levels are shown in the histograms (mean ± SD; n = 4). * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: To prepare the recombinant protein-coated plates, 1 µg of recombinant JAM-B (rJAM-B; 50464-M08H, SinoBiological, Beijing, China), recombinant JAM-C (rJAM-C; 50465-M08H, SinoBiological, Beijing, China), normal mouse IgG (12-371, Merck Millipore, Burlington, MA, USA) or Cellmatrix type I-A collagen (KP-2020, Nitta gelatin, Osaka, Japan) was placed in 12-well plates overnight at 4 °C.

Techniques: CRISPR, Western Blot, Cell Adhesion Assay, Quantitative RT-PCR, Expressing

Journal: Biomedicines

Article Title: Soluble JAM-C Ectodomain Serves as the Niche for Adipose-Derived Stromal/Stem Cells

doi: 10.3390/biomedicines9030278

Figure Lengend Snippet: Schematic model for the regulation of ADSC functions by soluble JAM-C. The soluble JAM-C (sJAM-C) deposited on the extracellular matrix interacts with JAM-B and partly with JAM-C on ADSC. sJAM-C/JAM-B signaling reaches the nucleus (dashed arrow) and stimulates cellular proliferation and expression of MSC markers (arrows). MSC, mesenchymal stromal/stem cell.

Article Snippet: To prepare the recombinant protein-coated plates, 1 µg of recombinant JAM-B (rJAM-B; 50464-M08H, SinoBiological, Beijing, China), recombinant JAM-C (rJAM-C; 50465-M08H, SinoBiological, Beijing, China), normal mouse IgG (12-371, Merck Millipore, Burlington, MA, USA) or Cellmatrix type I-A collagen (KP-2020, Nitta gelatin, Osaka, Japan) was placed in 12-well plates overnight at 4 °C.

Techniques: Expressing