Journal: The Journal of Neuroscience

Article Title: Embryonic Pericytes Promote Microglial Homeostasis and Their Effects on Neural Progenitors in the Developing Cerebral Cortex

doi: 10.1523/jneurosci.1201-21.2021

Figure Lengend Snippet: Figure 2. Administration of APB5 induced pericytes to undergo apoptosis. A, A schematic showing the strategy of in vivo adminis- tration of anti-PDGFRb antibodies (APB5). B, Representative immunostaining for CD31 (green), NG2 (magenta), and PDGFRb (top) or PDGFRa (bottom; cyan) of the E14 cerebral wall. In top figures, white arrowhead indicates NG21PDGFRb 1 pericytes and yellow arrowhead indicates NG21PDGFRb – OPCs. In bottom figures, white arrowhead indicates NG21PDGFRa– pericytes and yellow arrowhead indicates NG21PDGFRa1 OPCs. C, Immunostaining for NG2 (green), CD31 (magenta), and DAPI (blue) in the E14 cerebral wall. D, A graph comparing the proportion of pericyte coverage, which was calculated by dividing the merged area of NG2 and CD31 by the total CD311 area in the pallium (not including the meninges), between the saline-treated, isotype control antibody-treated, and APB5-treated brains (two-sided Steel–Dwass test; the average value of four sections from each animal is plotted; N = 6 mice; p= 1.000 and p=0.011; left to right). Data are presented as the mean value 6 SD. E, Immunostaining for Cl-Casp3 (green), CD31 (magenta), and DAPI (blue) in the cerebral wall of the mice treated with antibodies or saline. The white broken line indicates the ap- ical surface. Scale bar: 50mm (B) or 100mm (C, E).

Article Snippet: Single cells were stained with rat FITC rat anti-mouse CD31 mAb (1:200, catalog #102405, BioLegend, RRID: AB_312900) or rat FITC IgG2a isotype control Ab (1:200, catalog #400505, BioLegend, RRID: AB_2736919), rat anti-NG2 mAb (1:200, catalog #MAB6689, R&D Systems, RRID: AB_10890940), which was labeled by PE using an Ab-10 Rapid R-Phycoerythrin Labeling kit (LK34, Dojindo), or rat PE IgG1 isotype control Ab (1:200, catalog #400408, BioLegend, RRID: AB_ 326514), and rat BV421 anti-PDGFRa mAb (1:200, catalog #135923, BioLegend, RRID: AB_2814036) or rat BV421 IgG2a isotype control antibody (1:200, catalog #407117, BioLegend, RRID: AB_2687343) for 1 h on ice.

Techniques: In Vivo, Immunostaining, Saline, Control

Journal: The Journal of Neuroscience

Article Title: Embryonic Pericytes Promote Microglial Homeostasis and Their Effects on Neural Progenitors in the Developing Cerebral Cortex

doi: 10.1523/jneurosci.1201-21.2021

Figure Lengend Snippet: Figure 6. An in vitro coculture showed that pericytes promoted microglial proliferation. A, FACS analysis of pallial cells collected from E14 mice to detect PDGFRb , NG2, and PDGFRa expres- sion. B, Pericyte sorting strategy by FACS. NG21PDGFRa– cells were collected from E14 cerebral wall cells. C, A schematic showing the bilayer culture of CD11b1 microglia with or without NG21PDGFRa– cells (pericytes) using the Transwell insert. D, Immunostaining for CX3CR1 and Ki67 in microglia cultured with or without NG21PDGFRa cells (pericytes). E, A bar graph com- paring the density of CX3CR11 cells (two-sided Mann–Whitney U test; the average value of six fields per independent culture is plotted; N = 6 independent cultures; p = 0.002). F, A bar graph comparing the proportion of Ki671 cells among CX3CR11 cells (two-sided Mann–Whitney U test; the average value of six fields per independent culture is plotted; N = 6 independent cultures; p = 0.002). G, Immunostaining for CX3CR1 and BrdU in microglia cultured with or without NG21PDGFRa cells (pericytes). Scale bar: 20mm. H, A bar graph comparing the proportion of BrdU1 cells among CX3CR11 cells (two-sided Mann–Whitney U test; the average value of six fields per independent culture is plotted; N = 6 independent cultures; p = 0.002). Data are pre- sented as the mean value 6 SD. Scale bar: 50 mm (D) or 20mm (G).

Article Snippet: Single cells were stained with rat FITC rat anti-mouse CD31 mAb (1:200, catalog #102405, BioLegend, RRID: AB_312900) or rat FITC IgG2a isotype control Ab (1:200, catalog #400505, BioLegend, RRID: AB_2736919), rat anti-NG2 mAb (1:200, catalog #MAB6689, R&D Systems, RRID: AB_10890940), which was labeled by PE using an Ab-10 Rapid R-Phycoerythrin Labeling kit (LK34, Dojindo), or rat PE IgG1 isotype control Ab (1:200, catalog #400408, BioLegend, RRID: AB_ 326514), and rat BV421 anti-PDGFRa mAb (1:200, catalog #135923, BioLegend, RRID: AB_2814036) or rat BV421 IgG2a isotype control antibody (1:200, catalog #407117, BioLegend, RRID: AB_2687343) for 1 h on ice.

Techniques: In Vitro, Immunostaining, Cell Culture, MANN-WHITNEY

Journal: American Journal of Respiratory and Critical Care Medicine

Article Title: Nanoparticle Delivery of Proangiogenic Transcription Factors into the Neonatal Circulation Inhibits Alveolar Simplification Caused by Hyperoxia

doi: 10.1164/rccm.201906-1232OC

Figure Lengend Snippet: Polyethylenimine-(5) myristic acid/polyethylene glycol oleic acid/cholesterol (PEI600-MA5/PEG-OA/Cho) nanoparticles efficiently target endothelial cells in the neonatal lung. (A) Fluorescence-activated cell sorter (FACS) gating strategy to identify hematopoietic cells (Hema; CD45+CD31−), endothelial cells (Endo; CD31+CD45−CD326−), epithelial cells (Epi; CD326+CD45−CD31−), pericytes (Peric; NG2+PDGFRb+CD45−CD31−CD326−), and myofibroblasts (Myofibro; PDGFRa+CD45−CD31−CD326−). DyLight 650–labeled nanoparticles were delivered at Postnatal Day 2 (P2). FACS analysis of enzymatically digested lung tissue was performed at P5. (B and C) Dot plots show the presence of nanoparticles in different populations of pulmonary cells. Noninjected mice were used as control animals to identify cells containing nanoparticles. (D) Percentage of nanoparticle-targeted cells is shown among pericytes; myofibroblasts; and epithelial, endothelial, and hematopoietic cells (n = 4 mice per group). Error bars are mean ± SE. NG2 = melanoma-associated chondroitin sulfate proteoglycan 4; PDGFRα = platelet-derived growth factor receptor-α; PDGFRβ = platelet-derived growth factor receptor-β.

Article Snippet: Cells not expressing any of these markers (CD45 – CD31 – CD326 – ) were then evaluated for pericytes (PDGFRb + NG2 + CD45 – CD31 – CD326 – ) using PDGFRb (platelet-derived growth factor receptor-β) (CD140b) Ab (clone APB5; BioLegend) and NG2 (melanoma-associated chondroitin sulfate proteoglycan 4) Ab (clone 9.2.27; BD Biosciences).

Techniques: Fluorescence, Labeling, Derivative Assay