ccl21 levels  (Boster Bio)

Journal: eLife

Article Title: Inflammation rapidly recruits mammalian GMP and MDP from bone marrow into regional lymphatics

doi: 10.7554/eLife.66190

Figure Lengend Snippet: ( A ) Graph represents soluble Ccl19 chemokine in femoral or lymph node (LN) extracellular fluid and peripheral blood (PB) plasma after PBS (black lines) or lipopolysaccharide (LPS) (blue lines) administration at different circadian cycle times (zeitgeber time [ZT]4.5 [10.30 am, 0.5 hr after LPS administration], ZT5 [11 am, 1 hr after LPS administration], and ZT7 [1 pm, 3 hr after LPS administration]). ( B ) Strategy for in vivo neutralization of Ccr7 receptor or Ccl19 ligand by injections of anti-Ccr7 antibody or anti-Ccl19 antibody (50 μg/dose, two doses) into C57Bl/6 mice. One day after the last dose of antibodies, PBS or LPS was administered at ZT4 (10 am, time of LPS administration), and the myeloid progenitors-circulating cells from the LN and PB were measured by colony-forming unit (CFU) assay at ZT7 (1 pm, 3 hr after LPS administration). ( C, D ) Absolute number of progenitors present into LN from neutralized mice with anti-Ccl19/IgG (left graph) or anti-Ccr7/IgG2a (right graph) after PBS (black) or LPS (blue) administration as depicted in ( B ). ( E ) Generation of hematopoietic chimeric Ccl19 expressing (wild-type [WT]) or not (Ccl19 -/- ) mice and isolation of LNs after administration of PBS or LPS. ( F ) CFU content of LN from either WT or Ccl19 -/- hematopoietic chimeric animals treated with PBS or LPS. ( G ) Experimental design to analyze L-selectin dependence of femoral GMP migration to regional (or distant) LN after LPS administration. ( H, I ) Percentages of GFP + cells in LN after administration of an isotype control or anti-L-selectin antibodies. ( H ) Frequency of GFP + GMP cells in regional LN after administration of LPS was not modified by L-selectin blockade in vivo. ( I ) Inhibition of the migration of GFP + B-lymphocytes to regional LNs in mice pre-treated with anti-L-selectin antibody. In ( B ) and ( C ), LN were collected at ZT7 or 3 hr after LPS. Values represent mean ± SD of replicates in two or three independent experiments. *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: BM, plasma, and LN cells were isolated in PBS containing a protease inhibitor cocktail (Roche Diagnostics, Chicago, IL), and Ccl19/Ccl21 levels were determined by indirect sandwich of enzyme-linked immunosorbent assay (ELISA) following manufacturer’s instructions (R&D Systems, Minneapolis, MN).

Techniques: In Vivo, Neutralization, Colony-forming Unit Assay, Expressing, Isolation, Migration, Control, Modification, Inhibition

Journal: eLife

Article Title: Inflammation rapidly recruits mammalian GMP and MDP from bone marrow into regional lymphatics

doi: 10.7554/eLife.66190

Figure Lengend Snippet: ( A ) Ccl21 in femoral or LN extracellular fluid and peripheral blood (PB) plasma after PBS (left lines) or lipopolysaccharide (LPS) (right lines) administration at different circadian cycle times. ( B ) Ccl19 released into the supernatant from sorted wild-type (WT) and Traf6 -deficient LN T + cells, B + cells, and CD11b + myeloid cells after LPS stimulation in vitro. ( C–E ) Experimental schema of analysis of regional migration from chimeric femora of Ccr7-expressing GMP cells to bone marrow (BM) ( D ) and regional (inguinal and popliteal) LN ( E ). ( F, G ) Representative example of flow cytometry analysis ( F ) and mean fluorescence intensity (MFI) levels ( G ) of Ccr7 on GMPs from wild-type (WT) and Traf6-deficient mice in the presence of PBS (black and orange solid bars) or LPS (blue solid bar and orange mosaic bar). Values represent mean ± SD of two independent experiments. **p<0.01, ***p<0.001.

Article Snippet: BM, plasma, and LN cells were isolated in PBS containing a protease inhibitor cocktail (Roche Diagnostics, Chicago, IL), and Ccl19/Ccl21 levels were determined by indirect sandwich of enzyme-linked immunosorbent assay (ELISA) following manufacturer’s instructions (R&D Systems, Minneapolis, MN).

Techniques: In Vitro, Migration, Expressing, Flow Cytometry, Fluorescence

Journal: eLife

Article Title: Inflammation rapidly recruits mammalian GMP and MDP from bone marrow into regional lymphatics

doi: 10.7554/eLife.66190

Figure Lengend Snippet: Pharmacological regulation of lipopolysaccharide (LPS)/Toll-like receptor (TLR) signaling pathway. ( A ) Annexin-V binding to membrane phosphatidylserine (PS) on lymph node (LN) myeloid populations from wild-type (WT) (left mosaic bars) and Traf6 ∆∕∆ (right mosaic bars) (n = 4 mice per group) after PBS or LPS administration. LN suspension cells were stained for myeloid surface markers including annexin-V and analyzed by flow cytometry. ( B ) Transwell migration of low-density bone marrow (LDBM)-derived LK cells toward gradient generated by pre-treated LN cells with dimethylsulfoxide (DMSO) (solid bars) as vehicle control and inhibitors (mosaic bars) against Irak1/4 (right lined), Ubc13 (left lined), and IKK (white squares), and following TLR signaling pathway activation by PBS (black) or LPS (blue). ( C ) Analysis of SNAP23 phosphorylation (Ser95) in LN myeloid cells previously treated with DMSO (solid bar) as vehicle control, Irak1/4 (right lined mosaic bar), Ubc13 (left lined mosaic bar), or IKK (white squares mosaic bar) inhibitors. Values represent two independent experiments as mean ± SD of two independent experiments performed in triplicate. ( D ) Mean fluorescence intensity (MFI) quantification of pre-stored Ccl19 into LN-residing CD11b low /CD11c + cDC from non-manipulated mice by flow cytometry. Values represent mean ± SD of two or three independent experiments. p<0.05, **p<0.01, ***p<0.001.

Article Snippet: BM, plasma, and LN cells were isolated in PBS containing a protease inhibitor cocktail (Roche Diagnostics, Chicago, IL), and Ccl19/Ccl21 levels were determined by indirect sandwich of enzyme-linked immunosorbent assay (ELISA) following manufacturer’s instructions (R&D Systems, Minneapolis, MN).

Techniques: Binding Assay, Membrane, Suspension, Staining, Flow Cytometry, Migration, Derivative Assay, Generated, Control, Activation Assay, Fluorescence

Journal: eLife

Article Title: Inflammation rapidly recruits mammalian GMP and MDP from bone marrow into regional lymphatics

doi: 10.7554/eLife.66190

Figure Lengend Snippet: ( A ) Apoptosis (TUNELassay) of LK progenitors, Lin- and Lin + bone marrow (BM) cells. ( B ) Representative overlap histograms of phospho-SNAP23 (pSNAP23) into LN myeloid cells treated with dimethylsulfoxide (DMSO) (upper left), Irak1/4-inh (upper right), Ubc13-inh (lower left), and IKK-inh (lower right) and stimulated with PBS (red lines) or lipopolysaccharide (LPS) (blue lines). ( C ) Gating strategy to determine Ccl19 pre-stored into LN-residing myeloid cells basally. ( D ) Mean fluorescence intensity (MFI) quantification of pre-stored Ccl19 in pDC populations in basal conditions.

Article Snippet: BM, plasma, and LN cells were isolated in PBS containing a protease inhibitor cocktail (Roche Diagnostics, Chicago, IL), and Ccl19/Ccl21 levels were determined by indirect sandwich of enzyme-linked immunosorbent assay (ELISA) following manufacturer’s instructions (R&D Systems, Minneapolis, MN).

Techniques: Fluorescence

Journal: eLife

Article Title: Inflammation rapidly recruits mammalian GMP and MDP from bone marrow into regional lymphatics

doi: 10.7554/eLife.66190

Figure Lengend Snippet:

Article Snippet: BM, plasma, and LN cells were isolated in PBS containing a protease inhibitor cocktail (Roche Diagnostics, Chicago, IL), and Ccl19/Ccl21 levels were determined by indirect sandwich of enzyme-linked immunosorbent assay (ELISA) following manufacturer’s instructions (R&D Systems, Minneapolis, MN).

Techniques: Expressing, Knock-In, Control, Fluorescence, Sequencing, Purification, Extraction, Red Blood Cell Lysis, Flow Cytometry, Recombinant, Produced, Functional Assay, Transplantation Assay, Staining, Software, In Situ, Membrane, Transduction, Protease Inhibitor, Enzyme-linked Immunosorbent Assay, Activity Assay, Transport Assay, TUNEL Assay, End Labeling, Labeling