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Pneuma Cult™-Ex Plus Medium is a defined, serum- and BPE-free cell culture medium that supports more expansion of primary human airway epithelial cells at each passage, compared to other commercially available expansion media. This medium also supports at least two additional passages of cell expansion with better differentiation potential, defined as the ability to form a pseudostratified mucociliary epithelium at the air-liquid interface (ALI) using Pneuma Cult™-ALI Medium (Catalog #05001) or a cuboidal epithelium using Pneuma Cult™-ALI-S Medium (Catalog #05050). Pneuma Cult™-Ex Plus and either Pneuma Cult™-ALI or Pneuma Cult™-ALI-S constitute a fully integrated BPE-free culture system for in vitro human airway modeling. This robust and defined system is a valuable tool for basic respiratory research, toxicity studies, and drug development. Learn how to culture human airway epithelial cells at the ALI in our On-Demand Pulmonary Course or browse our Frequently Asked Questions (FAQs) about the ALI culture workflow using Pneuma Cult™.• A defined, serum- and BPE-free cell culture medium that delivers consistent performance • Pneuma Cult™-Ex Plus Medium supports more cell expansion at each passage compared to other commercially available expansion media • When used together with Pneuma Cult™-ALI Medium or Pneuma Cult™-ALI-S Medium, Pneuma Cult™-Ex Plus Medium supports better ALI differentiation potential even after extended passaging compared to other commercially available expansion media This product is designed for use in the following research area(s) as part of the highlighted workflow stage(s). Explore these workflows to learn more about the other products we offer to support each research area. Overview of the Pneuma Cult™ culture system Expansion of human bronchial epithelial cells (HBECs) in submerged culture is performed with Pneuma Cult™-Ex Plus or Pneuma Cult™-Ex. During the early “Expansion Phase” of the air-liquid interface (ALI) culture procedure, Pneuma Cult™-Ex Plus or Pneuma Cult™-Ex is applied to the apical and basal chambers. UTP: Calciumactivated Chloride channels (Ca CCs) activator. Upon reaching confluence, the culture is air-lifted by removing the culture medium from both chambers, and adding Pneuma Cult™-ALI to the basal chamber only. All ALI differentiation cultures were performed using Pneuma Cult™-ALI. Differentiation into a pseudostratified mucociliary epithelium is obtained following 21-28 days of incubation and can be maintained for more than one year. HBECs cultured in Pneuma Cult™-Ex Plus have a faster expansion rate compared to those cultured in Pneuma Cult™-Ex and Bronchial Epithelial Growth Media Commercially available, cryopreserved P1 HBECs were seeded into Pneuma Cult™-Ex Plus, Pneuma Cult™-Ex, or Bronchial Epithelial Growth Media. INTRODUCTION One method for assessing the in vitro response to CFTR-modulating compounds is by analysis of epithelial monolayers in an Ussing chamber, where the apical and basolateral surfaces are isolated and the potential difference, short-circuit current, and transepithelial resistance can be monitored. Cells cultured in Pneuma Cult™-Ex Plus have a significantly higher proliferation rate over 9 passages compared to those maintained in either control medium (n=6). Representative morphology of HBECs Representative live culture images for P4 HBECs cultured in Pneuma Cult™-Ex Plus, Pneuma Cult™-Ex, or Bronchial Epithelial Growth Media. HBECs cultured in Pneuma Cult™-Ex Plus maintain widespread expression of the basal cell markers CD49f and CD271 Immunocytochemistry detection of basal cell markers - CD49f (A, B, and C) and CD271 (D, E, and F) - for P4 HBECs cultured in Pneuma Cult™-Ex Plus (A and D), Pneuma Cult™-Ex (B and E), and Bronchial Epithelial Growth Media (C and F). cells P4 HBECs cultured in Pneuma Cult™-Ex Plus (A), Pneuma Cult™-Ex (B), and Bronchial Epithelial Growth Media (C) were characterized by flow cytometry to detect expression of the basal cell markers CD49f and CD271. Electrophysiological characterization of differentiated HBECs (P4) that were expanded in Pneuma Cult™-Ex Plus, Pneuma Cult™-Ex, and Bronchial Epithelial Growth Media Transepithelial electrical resistance (TEER) (A) and representative characterization of the ion channel activities (B) for ALI cultures at 28 days post air-lift using HBECs expanded in Pneuma Cult™-Ex Plus, Pneuma Cult™-Ex, or Bronchial Epithelial Growth Media. The effect of a chloride ion gradient across airway epithelia on transepithelial chloride transport and the magnitude of CFTR modulator efficacy were examined. Cells cultured in Pneuma Cult™-Ex Plus (A) are smaller and more tightly packed than those cultured in Pneuma Cult™-Ex (B) or Bronchial Epithelial Growth Media (C). HBECs cultured in Pneuma Cult™-Ex Plus (A) have a higher proportion of cells coexpressing CD49f and CD271, compared to those cultured in Pneuma Cult™-Ex (B) and Bronchial Epithelial Growth Media (C). HBECs cultured in Pneuma Cult™-Ex Plus differentiate into a pseudostratified mucociliary epithelium at later passages with the use of Pneuma Cult™-ALI P4 HBECs were seeded and passaged using Pneuma Cult™-Ex Plus, Pneuma Cult™-Ex, or Bronchial Epithelial Growth Media, followed by ALI differentiation at each passage (P5-8) with the use of Pneuma Cult™-ALI. METHODS CFTR-mediated changes in the potential difference and transepithelial currents of primary human nasal epithelial cell cultures were quantified in Ussing chambers with either symmetrical solutions or reduced chloride solutions in the apical chamber. The ALI cultures at 28 days post air-lift were fixed and stained with antibodies for cilia marker AC-tubulin (red) and the goblet cell marker Muc5AC (green). CFTR activity in homozygous F508del CFTR epithelia was rescued by treatment with VX-661, C4/C18, 4-phenylbutyrate (4-PBA) for 24 hr at 37°C or by incubation at 29°C for 48 hr. RESULTS Imposing a chloride gradient increased CFTR-mediated and Ca CC-mediated ion transport. Treatment of F508del CFTR homozygous cells with CFTR modulating compounds increased CFTR activity, which was significantly more evident in the presence of a chloride gradient. This observation was recapitulated with temperature-mediated F508del CFTR correction. CONCLUSIONS Imposing a chloride gradient during Ussing chamber measurements resulted in increased CFTR-mediated ion transport in expanded non-CF and F508del CFTR homozygous epithelia. In F508del CFTR homozygous epithelia, the magnitude of response to CFTR modulating compounds or low temperature was greater when assayed with a chloride gradient compared to symmetrical chloride, resulting in an apparent increase in measured efficacy. Future work may direct which methodologies utilized to quantify CFTR modulator response in vitro are most appropriate for the estimation of in vivo efficacy. Air-liquid interface (ALI) culture of nasal epithelial cells is a valuable tool in the diagnosis and research of primary ciliary dyskinesia (PCD). Ex vivo samples often display secondary dyskinesia from cell damage during sampling, infection or inflammation confounding PCD diagnostic results. ALI culture enables regeneration of healthy cilia facilitating differentiation of primary from secondary ciliary dyskinesia. We describe a revised ALI culture method adopted from April 2018 across three collaborating PCD diagnostic sites, including current University Hospital Southampton COVID-19 risk mitigation measures, and present results. Two hundred and forty nasal epithelial cell samples were seeded for ALI culture and 199 (82.9) were ciliated. Fifty-four of 83 (63.9) ex vivo samples which were originally equivocal or insufficient provided diagnostic information following in vitro culture. Surplus basal epithelial cells from 181 nasal brushing samples were frozen in liquid nitrogen; 39 samples were ALI-cultured after cryostorage and all ciliated. The ciliary beat patterns of ex vivo samples (by high-speed video microscopy) were recapitulated, scanning electron microscopy demonstrated excellent ciliation, and cilia could be immuno-fluorescently labelled (anti-alpha-tubulin and anti-RSPH4a) in representative cases that were ALI-cultured after cryostorage. In summary, our ALI culture protocol provides high ciliation rates across three centres, minimising patient recall for repeat brushing biopsies and improving diagnostic certainty. Cryostorage of surplus diagnostic samples was successful, facilitating PCD research. Idiopathic pulmonary fibrosis (IPF) is a fatal disease of unknown cause that is characterized by progressive fibrotic lung remodeling. An abnormal emergence of airway epithelial-like cells within the alveolar compartments of the lung, herein termed bronchiolization, is often observed in IPF. However, the origin of this dysfunctional distal lung epithelium remains unknown due to a lack of suitable human model systems. In this study, we established a human induced pluripotent stem cell (i PSC)-derived air-liquid interface (ALI) model of alveolar epithelial type II (ATII)-like cell differentiation that allows us to investigate alveolar epithelial progenitor cell differentiation in vitro. We treated this system with an IPF-relevant cocktail (IPF-RC) to mimic the pro-fibrotic cytokine milieu present in IPF lungs. Stimulation with IPF-RC during differentiation increases secretion of IPF biomarkers and RNA sequencing (RNA-seq) of these cultures reveals significant overlap with human IPF patient data. IPF-RC treatment further impairs ATII differentiation by driving a shift toward an airway epithelial-like expression signature, providing evidence that a pro-fibrotic cytokine environment can influence the proximo-distal differentiation pattern of human lung epithelial cells. In conclusion, we show for the first time, the establishment of a human model system that recapitulates aspects of IPF-associated bronchiolization of the lung epithelium in vitro. Shifts in cellular metabolic phenotypes have the potential to cause disease-driving processes in respiratory disease. The respiratory epithelium is particularly susceptible to metabolic shifts in disease, but our understanding of these processes is limited by the incompatibility of the technology required to measure metabolism in real-time with the cell culture platforms used to generate differentiated respiratory epithelial cell types. Thus, to date, our understanding of respiratory epithelial metabolism has been restricted to that of basal epithelial cells in submerged culture, or via indirect end point metabolomics readouts in lung tissue. Here we present a novel methodology using the widely available Seahorse Analyzer platform to monitor real-time changes in the cellular metabolism of fully differentiated primary human airway epithelial cells grown at air-liquid interface (ALI). We show increased glycolytic, but not mitochondrial, ATP production rates in response to physiologically relevant increases in glucose availability. We also show that pharmacological inhibition of lactate dehydrogenase is able to reduce glucose-induced shifts toward aerobic glycolysis. This method is timely given the recent advances in our understanding of new respiratory epithelial subtypes that can only be observed in vitro through culture at ALI and will open new avenues to measure real-time metabolic changes in healthy and diseased respiratory epithelium, and in turn the potential for the development of novel therapeutics targeting metabolic-driven disease phenotypes. Coronavirus disease 2019 (COVID-19) is caused by SARS-Co V-2, an emerging virus that utilizes host proteins ACE2 and TMPRSS2 as entry factors. Understanding the factors affecting the pattern and levels of expression of these genes is important for deeper understanding of SARS-Co V-2 tropism and pathogenesis. Here we explore the role of genetics and co-expression networks in regulating these genes in the airway, through the analysis of nasal airway transcriptome data from 695 children. We identify expression quantitative trait loci for both ACE2 and TMPRSS2, that vary in frequency across world populations. We find TMPRSS2 is part of a mucus secretory network, highly upregulated by type 2 (T2) inflammation through the action of interleukin-13, and that the interferon response to respiratory viruses highly upregulates ACE2 expression. IL-13 and virus infection mediated effects on ACE2 expression were also observed at the protein level in the airway epithelium. Finally, we define airway responses to common coronavirus infections in children, finding that these infections generate host responses similar to other viral species, including upregulation of IL6 and ACE2. Our results reveal possible mechanisms influencing SARS-Co V-2 infectivity and COVID-19 clinical outcomes. BACKGROUND Taking into consideration a recent surge of a lung injury condition associated with electronic cigarette use, we devised an in vitro model of sub-chronic exposure of human bronchial epithelial cells (HBECs) in air-liquid interface, to determine deterioration of epithelial cell barrier from sub-chronic exposure to cigarette smoke (CS), e-cigarette aerosol (EC), and tobacco waterpipe exposures (TW). METHODS Products analyzed include commercially available e-liquid, with 0 or 1.2 concentration of nicotine, tobacco blend (shisha), and reference-grade cigarette (3R4F). In one set of experiments, HBECs were exposed to EC (0 and 1.2), CS or control air for 10 days using 1 cigarette/day. In the second set of experiments, exposure of pseudostratified primary epithelial tissue to TW or control air exposure was performed 1-h/day, every other day, until 3 exposures were performed. After 16-18 h of last exposure, we investigated barrier function/structural integrity of the epithelial monolayer with fluorescein isothiocyanate-dextran flux assay (FITC-Dextran), measurements of trans-electrical epithelial resistance (TEER), assessment of the percentage of moving cilia, cilia beat frequency (CBF), cell motion, and quantification of E-cadherin gene expression by reverse-transcription quantitative polymerase chain reaction (RT-q PCR). RESULTS When compared to air control, CS increased fluorescence (FITC-Dextran assay) by 5.6 times, whereby CS and EC (1.2) reduced TEER to 49 and 60 respectively. CS and EC (1.2) exposure reduced CBF to 62 and 59, and cilia moving to 47 and 52, respectively, when compared to control air. CS and EC (1.2) increased cell velocity compared to air control by 2.5 and 2.6 times, respectively. The expression of E-cadherin reduced to 39 of control air levels by CS exposure shows an insight into a plausible molecular mechanism. Altogether, EC (0) and TW exposures resulted in more moderate decreases in epithelial integrity, while EC (1.2) substantially decreased airway epithelial barrier function comparable with CS exposure. CONCLUSIONS The results support a toxic effect of sub-chronic exposure to EC (1.2) as evident by disruption of the bronchial epithelial cell barrier integrity, whereas further research is needed to address the molecular mechanism of this observation as well as TW and EC (0) toxicity in chronic exposures. Legal Statement: This product was developed under a license to intellectual property owned or controlled by Propagenix, Inc. This product is sold for research use only (which includes pre-clinical research) under a non-transferable, limited-use license. Purchase of this product does not include the right to sell, use or otherwise transfer this product for commercial purposes or clinical use. Purchasers wishing to use the product for purposes other than research use should contact Propagenix, Inc. Quality Statement: PRODUCTS ARE FOR RESEARCH USE ONLY AND NOT INTENDED FOR HUMAN OR ANIMAL DIAGNOSTIC OR THERAPEUTIC USES UNLESS OTHERWISE STATED. FOR ADDITIONAL INFORMATION ON QUALITY AT STEMCELL, REFER TO WWW. I’m sharing tried-and-true recipes I’ve made dozens (even hundreds) of times, because I’ve never tasted anything better. Monday I posted the absolute best yeast roll recipe I’ve ever made, and today I’m sharing The BEST Sugar Cookie Recipe Ever. Learn How To Make The BEST Sugar Cookies With This Fun & Easy Homemade Sugar Cookies Recipe! The Best Sugar Cookie Recipe is ultra-light, ever so crispy around the edges, glistening with sugar crystals, and packed with a sweet vanilla-wheat flavor. And There’s something spectacular about a simple, yet perfect, soft sugar cookie. These cookies put all other sugar cookies to shame. Today I’m going to show you how to make sugar cookies that will make your head spin. Friends, this is the end-all of sugar cookies recipes. I bake “The Best Sugar Cookie Recipe” for holidays and parties regularly, and have used this recipe as a base for all sorts of sugar cookie variations. I’ve been asked for The Best Sugar Cookie Recipe more times than I can count, always with a statement like, “These are just sugar cookies, right? I’ve got to have the recipe.” I found the base recipe at least a decade ago in an old version of Cook’s Illustrated’s Best Recipe Cookbook. Over the years, I’ve tweaked it just a tad, so that now I have the Please note: The Best Sugar Cookie Recipe is not a roll-out cookie recipe for cookie cutters. If you’re looking for a roll-and-cut cookie recipe, use this one. The extra egg yolk and flour make the cookies slightly sturdier, so they can handle the abuse. However, these sugar cookies This goes back to mismeasuring flour. Try using vegan or plant-based butter, vegetable shortening, or margarine, instead of unsalted (dairy) butter. You can keep the unbaked dough, or baked cookies without any frosting, in the freezer for up to 3 months, as long as it is contained in an airtight container. Most likely, your flour was too packed into the measuring cups, so there is too much flour in the recipe. We’ve made this recipe with several brands of 1-to-1 gluten-free baking flour, with great success. Yet the cookies are still light, soft in the middle, and crispy around the edges. If you are wanting to freeze baked cookies, make sure to separate them with layers of parchment paper so they don’t stick together. As mentioned above, this recipe produces cookies that are too delicate to roll and cut. You can thaw them out in the fridge or at room temperature. Allow the excess to dip off, then place them back on the parchment paper to dry. You can use any sugar substitute, as long as it is measured in a 1-to-1 ratio with granulated sugar. BOURBON GLAZE: Once the cookies are cool, whisk 1 tablespoon of heavy cream, 1 teaspoon vanilla extract, and 1 teaspoon bourbon together. Miles “Pudge” Halter is done with his safe life at home. His whole life has been one big non-event, and his obsession with famous last words has only made him crave “the Great Perhaps” even more (Francois Rabelais, poet). He heads off to the sometimes crazy and anything-but-boring world of Culver Creek Boarding School, and his life becomes the opposite of safe. Miles “Pudge” Halter is done with his safe life at home. His whole life has been one big non-event, and his obsession with famous last words has only made him crave “the Great Perhaps” even more (Francois Rabelais, poet). He heads off to the sometimes crazy and anything-but-boring world of Culver Creek Boarding School, and his life becomes the opposite of safe. The gorgeous, clever, funny, sexy, self-destructive, screwed up, and utterly fascinating Alaska Young. She pulls Pudge into her world, launches him into the Great Perhaps, and steals his heart. Broccoli and Cheese Stuffed Chicken are filled with cheddar and broccoli, then rolled up, breaded and baked in the oven or air fryer! I make it with fresh broccoli, but leftover or frozen broccoli can also be used. There’s so many different ways to make stuffed chicken. This broccoli and cheese version is a favorite, but we also love Chicken Cordon Bleu, Zucchini and Mozzarella Stuffed Chicken and Chicken Rollatini Parmesan, to name a few. Broccoli and Cheese Stuffed Chicken, broccoli and cheese stuffed chicken breast, broccoli and cheese stuffed chicken breast recipe, easy broccoli and cheese stuffed chicken, stuffed broccoli and cheese chicken breast Hi, I’m Gina Homolka, a busy mom of two girls, author and recipe developer here at My food philosophy is to eat seasonal, whole foods and maintain good portion control (everything in moderation! If you want to enjoy movies and games these days you must have 6 channel sound system because all latest games and DVD movies are optimized and programmed to give a natural surround sound experience. If you have 5.1 speakers and don’t know how to connect it to your computer we’ll show you some easy steps on how to connect your speakers to computer the right way. The number 5.1 means that you have six speakers arranged in a way that two are on the front, two are on the rear, one on the front center which is known as voice channel and one subwoofer to provide bass. So you have five speakers and one subwoofer, this makes 6 channel sound system 5.1. There are two kinds of 6-channel surround speakers: analog and digital. Analog speakers are cheaper compared to digital and can be installed on any PC while digital speakers require that you have a SPDIF output on your PC. If your PC has SPDIF output than you might want to use a digital system. The steps described here will be just the same, but instead of connecting your PC to a home theater receiver you will be connecting it to a digital 6-channel speakers system. Generally in systems all the cables from the speakers are connected to the subwoofer which comes with a set of wires to connect, that includes : a power cord to connect the system to the AC outlet, a power connector to feed the subwoofer and the subwoofer wires. Your system should come with at least three set of cables for connecting your speaker system to your PC. These cables are usually colored to help with the installation. You should connect the cables matching the colors on the jacks located on the subwoofer. After connecting all these cable, its time to connect the rest of the cables like power, subwoofer and speakers. This is very easy if you pay attention to the subwoofer connection then you’d be able to follow the polarity that is written on the connectors. In the end, all your cables would be connected perfectly. If your computer doesn’t have these two extra outputs: rear out and center/subwoofer out, it’s a disadvantage, you’d be able to connect your 5.1 anyway but you would require to use the line in and mic-in plugs as an alternative. Of course there is a really big disadvantage in using this configuration: you cannot use your microphone or line in inputs and your 5.1 speaker system at the same time. This is the reason that you should buy a motherboard with 5.1 system built in. After every single connection has been made, its time to setup audio configuration in operating system. Since we had Realtec drivers on our PC, Double clicking it opened up Speaker Configuration tab. In various options you should select 6 channel mode for 5.1 speaker output and check Only surround-kit if your PC has the separated rear and center/subwoofer outputs and leave it if you don’t. part may be reproduced in any form without explicit written permission.