![]() However, these are mostly collector cars that will rarely be driven or exposed to the sun for prolonged periods of time.įor this reason, we do sell some furniture-grade leathers. When you’re doing one-off builds with some extreme angles and compound curves, you need to have some flexibility. Some of our hot rod and custom car upholsterers prefer to work with furniture leather because of its elasticity. ![]() There are applications where an automotive leather just won’t work because it’s too stiff or the wrong look. Is there any case where using furniture leather to upholster cars is a good idea? This is especially true with waxed and oiled leathers, or aniline leathers, as they have minimal or no urethane top coat for protection. The other factor is that the colors can fade because there isn’t enough UV resistance in the leather. What are the possible negative consequences of using furniture leather in cars?įurniture leather can be too soft for a car’s interior, and overtime the leather can stretch and become loose without rebound. I have seen cases where excessive moisturizing has altered the moisture content inside the leather hide, which causes the leather fibers to lose their strength and the interior becomes very loose, and in some cases dashboards bubble up or shrink. Occasional moisturizers are not going to hurt, but their benefits are also debatable. When lotions are applied to it, it’s really just sitting on the surface and then rubbing off on clothing. ![]() The proper amount of ingredients have been added to make the leather last as long as possible, and it has been tested thoroughly.Īuto leather has a pigmented and urethane top coat to seal it. The leather has been tanned to preserve it. Therefore, I feel that adding a lot of leather protectants and lotions is unnecessary. Is that also true of automotive leather?Īutomotive-grade leather by nature has a lot of protectant in it. There are also extra additives in the pigment dyes for durability and abrasion resistance and to minimize off-gassing (the windshield fog that we all wipe away).įurniture leather needs a lot of upkeep. In automotive leather, there is more UV protectant to block out the sun’s rays. There are differences in the surface finish. Is automotive leather chemically treated differently than furniture leather? This creates a leather that is more resistant to stretching and bagging. In automotive leather, durability and long life is needed so less fat liquors are used to create a stronger hide. ![]() Furniture leather uses a lot of fat liquor to make the skin fibers elastic and soft, which creates a supple leather. The primary difference is in the tanning process. What is the primary difference between automotive and furniture leather? We recently spoke with Aaron about the differences between automotive and furniture leather, the pros and cons of each, and how his team of experts can pretty much match any color out there. Meanwhile, furniture leather is softer and available in many different looks and finishes, but is susceptible to the elements and unsightly stretching.īut don’t just take our word for it - listen to Aaron Laithwaite, founder and CEO of Hydes Leather, the auto upholstery industry’s premier supplier of OEM automotive-grade leather. For example, automotive leather is stiffer and designed to withstand UV rays and abrasions, but unavailable in distressed or naked finishes. Auto trimmers should be aware of the significant differences between automotive- and furniture-grade leather before choosing a hide to upholster a client’s car.īoth types of leather offer benefits and drawbacks that must be taken into account.
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![]() It's too early to asses the evolution of the game and knowing if it'll become good or not but the basis are already there. Following what is happening is difficult. Your mission is help her grow her failing hotel’s business before it goes into receivership. No other sex tube is more popular and features more Fresh Girl scenes than Pornhub Browse through our impressive selection of porn videos in HD quality on any device you own. Discover the growing collection of high quality Most Relevant XXX movies and clips. ![]() Screen: COFreshGirls-0.5.5-pc.zip COFreshGirls-0.5.5-pc. About:Sunshine Love Version 0.0.6 Update You play the main character and must travel from his home to Sunshine Bay at the beginning of the game.He’s there to help his sister run the Sunshine Bay Bed and Breakfast Hotel. Watch Fresh Girl porn videos for free, here on. The characters and the background are well done.Īnd the main problem is the english. Version: 0.5.5 OS: Windows Language: English Size: 768 MB About This Game: The main character is on his first year of college help him in his journey with his decisions getting in troubles, romances, studies, sex and the basketball team. Also, is the MC that frustated sexually to have sex with every woman he encounters? I know it's a porn game but some feelings and romance (what, it's in the tags but I didn't see any) don't do anything bad. For a dating sim game, it's really strange not to develop the relationship with potential partners (targets but I don't like that term) before having some action. The first night you're already invited to a truth or dare game by only girls that are good with showing their body and, when you wake up after drinking too much, the only thing you think is fucking the closest girl to you. You go to the library and there's already an exhibitionnist you can fuck. The characters are already numerous and different but not enough male characters for the basketball team. 1 Overview: The main character is on his first year of college help him in his journey with his decisions. ![]() The worldbuilding is just a school so limited. “All I know is there’s a bunch of gunfire. He went to the dentist or somewhere and came home,” Payne replied. “OK, OK, and nobody saw anything prior to this?,” the operator asked. Cries could be heard in the call’s background. Payne told the dispatcher she heard gunfire and found her grandson lying in a doorway. “I don’t know if he’s OK or not ‘cause, he’s still out there.” “My grandson just got shot in the back when he come in the house,” Payne told the operator. Sharon Payne, Goodson’s grandmother, told the operator that people were preventing her from seeing her grandson, according to the two-minute recording obtained by CNN on Wednesday from the Columbus Division of Police. Authorities are investigating what happened that day and say Goodson was brandishing a gun before he was shot. Goodson, 23, was shot on Friday by a 17-year veteran of the Franklin County Sheriff’s Office, identified as deputy Jason Meade. In June, Franklin County Prosecutor Gary Tyack appointed two outside prosecutors to investigate since the county prosecutor’s office serves as legal counsel to the sheriff’s office and anticipates having to defend the county and the law enforcement agency in this case.ĮDITOR'S NOTE: The Associated Press contributed to this report.Casey Goodson’s grandmother told a 911 dispatcher that he had been shot in the back and she wasn’t sure if he was breathing moments after his fatal encounter with a law enforcement officer at his Ohio home, according to an audio recording of the call. As I've said from the very beginning, I pray for everyone involved in this tragedy.īaldwin had previously said the autopsy did not "provide all of the facts needed,” and that he will wait until the criminal investigation is complete before pursuing any disciplinary action against Meade. This office has a professional obligation to do everything in its power to ensure the community and our deputies are kept safe. It's vital to maintain that trust, which is why I've tasked members of my staff to review the facts from the independent investigation when we're able to fully access them and determine how this agency can best learn from this tragedy. As law enforcement officers we must meet this higher standard because of the immense trust we ask the community to place in us. Prior to retirement, he had been on administrative leave since the deadly shooting.įollowing Meade's indictment, Franklin County Sheriff Dallas Baldwin released the following statement:Īfter the grand jury indictment today, I've reminded my staff that while everyone is presumed innocent until proven guilty, the standards for being a Franklin County Sheriff's Deputy must be even higher than that of our criminal justice system. The manner of death was "homicide." The full coroner's report can be read here.Īfter 17 years with the sheriff's office, Meade retired July 2 on disability. She determined the cause of death was "gunshot wounds of the torso." She said "torso" is a term to define the midsection of the body, which includes front and back. ![]() Five of the bullets entered Goodson's back and one entered his buttock, according to Ortiz. Anahi Ortiz said in March 2021 that Goodson was shot a total of six times. ![]() ![]() There is no body camera footage of the actual shooting, as Franklin County Sheriff's task force officers are not issued body camerasįranklin County Coroner Dr. was not the person the task force was searching for. Meade was working with the US Marshal’s Southern Ohio Fugitive Task Force when he shot Goodson Jr. The incident happened in Northeast Columbus, just off Ferris Road. Goodson Jr., 23, was shot and killed by Meade on December 4, 2020. The charges were filed in Franklin County Court. Jason Meade was indicted Thursday on two charges of murder and one count of reckless homicide. COLUMBUS, Ohio ( WSYX) - After nearly a year of investigative work, a former Ohio sheriff's deputy has been charged with the murder of Casey Goodson Jr. ![]() These developments have made Timeless Treasures a unique one stop resource with expanded creative offerings for quilt shops and quilting enthusiasts around the world. In 2019 Timeless acquired Marcus Fabrics to offer its customers traditional and reproduction fabrics as well. In 2008, the Dear Stella brand was introduced to serve a growing contemporary and modern aesthetic. Timeless Treasures continues to evolve and meet an ever changing market demand. Red and Black, Buffalo Check, 100 Cotton, Quilter’s Fabric, Christmas Cabin, Timeless Treasures, C5784-Red, by The Yard. The line includes quilting collections, blenders, batiks, flannels and more. Timeless Treasures is still recognized for its unique novelty prints, bringing fun and playful designs to life. Today, Timeless Treasures is a leading fabric source for quilters- recognized for its diverse line of high quality, innovative and beautifully detailed designs. Timeless Treasures is a leading fabric source for quilters- recognized for its diverse line of high quality, innovative and beautifully detailed designs. In the early 1990's, Timeless introduced its first cotton fabric collection designed for the quilting industry and was initially known for its fun and creative novelties. Designing patterns is just so interesting and challenging.Timeless Treasures Fabrics was established in 1968 as a fashion fabric supplier in the heart of Soho, New York City. Pair a Basic, simple design with a more intricate pattern as you piece together your next quilt and see how Timeless Treasures balances their designs, colors. ![]() I have been a quilter for 45 years and a longarm quilter for 22 years. I sold my very first pattern years ago through Nancy’s Notions, and a ton of them were sold. Thanks for keeping Nancy’s Notions active. Thankful Fall Inclination Quit Kit by Timeless Treasures. Click here to find the kit: KIT: COSMIC BUTTERFLIES Large Throw/twin Size Quilt 63 – Etsy Arabella, Complete Quilt Kit featuring Tonga Batiks and pattern by Wing And A Prayer. Details 100 Cotton Quilting Fabric by the yard.Premium 44 inch wide cotton quilting fabric. Labels: Free Quilting Pattern, Quilting, Tonga Focus Frequency Quilt. Email This BlogThis Share to Twitter Share to Facebook Share to Pinterest. Full Post: Tonga Focus Frequency Quilt Pattern. Windham Fabrics ABCs in Bloom Kelly Angelovic Picnic Blanket White. This Tonga Focus Frequency Quilt designed by Osie Lebowitz of Timeless Treasures fabrics, The Pattern is available for free. I have found it to be the very best sealer and the. Premium 44 inch wide cotton quilting fabric. Royal Plume Pattern - Free Pattern Download. I designed the pattern Barn Quilts for the amazing Landscape Stripes collection by Timeless Treasures. I sell the patterns wholesale to quilt shops, and I also sell the patterns and kits online in my Etsy shop. Royal Plume Pattern - Free Pattern Download-Timeless Treasures-My Favorite Quilt Store. Timeless Treasures is a leading fabric source for quilters due to their assorted line of high quality and innovative designs. I’m not doing a pattern for that one but it’s beautiful also. Quilt Doodle Designs, Quilt Gate, Quilt Lily Designs, Quilt Poetry, Quilted Frog, Quilted Garden Designs, Quilters Clutter, Quilting Creations, Quilting. ![]() The second picture is a king size quilt that I designed for a friend who likes big quilts. The first picture is a large throw quilt, and the one I have designed a pattern for and made up some kits. The vibrant colors are absolutely gorgeous! I design patterns and I love butterflies, so I thought this was perfect for me. Hancock’s of Paducah is offering a kit if you’d like to make Panel Play Bows. Fat Quarter Shop features the latest quilting fabric collections including I Love You To The Moon and Back by Timeless Treasures Fabrics. The first pattern is called Panel Play Bows, and it features the Silent Night collection. I was fascinated with Timeless Treasures new Cosmic Butterflies fabrics and placed an order with my wholesale company early. JPatterns I often design patterns for fabric companies, and recently I designed two new patterns for new fabric collections by Timeless Treasures. ![]() I love the idea of Quilt & Tell, and I’m excited to tell you about my latest project. But pets are prohibited on Virginia’s portion of the island – even if they stay inside your vehicle.įortunately, you’ll find plenty to do on Maryland’s portion of the island! Most of the Assateague Island National Seashore and Assateague State Park – Maryland’s only oceanfront park – are located there. Explore the most popular beach trails in Assateague Island National Seashore with hand-curated trail maps and driving directions as well as detailed reviews. Sites have a picnic table and a fire ring. Oceanside Walk-in: Tent-only sites, located 100-200 feet from centralized parking areas. A casino & outlet shopping are within minutes. Assateague Island, home of the infamous wild ponies is just 10 miles away. Assateague Island is a beautiful barrier island, administrated by park services, permanently uninhabited, and most famous for its charismatic wild ponies. ![]() ![]() Beach, boardwalk, rides, amusements, shopping, restaurants, water sports, nightlife are all within walking distance. Assateague Island is a 37-mile-long barrier island with two-thirds in Maryland and one-third in Virginia. Sites have a picnic table and a fire ring. Located just 1/2 block from the Beach & Boardwalk, the Madison Beach Motel has rooms and renovated apartments. Assateague Beach is just outside the Chincoteague National Wildlife Refuge on Assateague Island. Virginia’s southern third of the island is home to the Assateague Lighthouse and the Chincoteague National Wildlife Refuge. Assateague Island National Seashore Campgrounds: Oceanside Drive-in: Tents, trailers and recreational vehicles (no hookups). With miles and miles of beach, great hiking trails, paved bike paths, and canoe and kayak launching areas, this is a place where you can really enjoy nature! Assateague Island – The Lay Of The LandĪssateague Island is located just 150 miles east of Washington, DC and is divided by the Maryland-Virginia state line. Assateague Island is a great choice for a laid-back dog friendly beach vacation on the east coast. Assateague Island, a 37-mile long barrier island located off the coast of Maryland and Virginia, is most known for the more than 300 wild ponies who wander the beaches. ![]() Some however are lucky enough to move up and they deserve it with all the hard work you have to put up with at this company, Lastly they forced lab techs to work the holidays and only told them ONE WEEK IN ADVANCE and threatened their jobs if they didn't come in.l However not one person in the office had to work the holidays. Details & Directions to 520 Larkfield Road East Northport. 520 Larkfield Road East Northport, NY 110. They also promise people they will be moving up in departments just to drag out them working there. Get FREE flu shots with most insurance at CVS in East Northport, New York. They expect certain people to do just about every position with out pay compensation. And then theirs upper management which is the biggest problem. They are willing to go above and beyond to help one another out. 361 Larkfield Rd, East Northport, NY 11731-2907 +1 63 Website Menu. The collecting team as in the associates molecular and specimen processors, are also a delight to work for. 18 reviews 9 of 46 Restaurants in East Northport - Cafe. However the supervisors are wonderful and do their best to help. The work place is unorganized and irrational. We employ state-of-the art tools and procedures to monitor medications, as well as detect the presence of illicit drugs. Also be aware the managers have disgusting attitudes toward lab techs, (Molecular and specimen processing). Acutis is a company at the leading edge of clinical toxicology. I have never seen such horrible communication in a "well established" company. Read 72 customer reviews of Acutis Diagnostics, one of the best Laboratory Testing businesses at 6108, 728 Larkfield Rd, East Northport, NY 11731 United. Something changes everyday and your just expected to know about it there are times the supervisors are unaware of the changes made that day. ![]() 577 Larkfield Road East Northport, NY 11731 US. 1 Rite Aid Store in East Northport, New York. They act like they care but they honestly could care less about their employees. Rapid Diagnostic Testing chevron right Vaccine Central chevron right. ![]() Other Source Control systems may be supported by using specific plugins. (Click on the links for specific instructions for each Source Control system) CVS, Git, Mercurial, Perforce and Subversion are supported natively. You can also import projects from a Source Control system if you use it. Depending on the type of project, you may need to change the "natures" associated with the project.Name your project appropriately and click OK.Expand the local filesystem node to the folder in question.You will see the project in your workspace, but the files live on disk in the former spot. Promoting a folder to a project allow you to take any folder on disk and convert it into a project. ![]() You will want to choose Web in almost every case, plus the type of your project (PHP, Rails, etc) In the Directory text field, browse to the folder that contains your Dreamweaver or HomeSite project.To import an existing project into Studio: CC BY-SA 3.0 Creative Commons Attribution-Share Alike 3.You can import an existing set of files into Aptana Studio by creating a new project and pointing Aptana Studio to the home directory for your existing project. It comes with modules that let you use it to create programs with Adobe Air technology and to create iPhone utilities. This licensing tag was added to this file as part of the GFDL licensing update. Aptana Studio is an integrated development environment (IDE) for building dynamic web applications that use PHP, Ruby, Ruby on Rails, and Python. share alike – If you remix, transform, or build upon the material, you must distribute your contributions under the same or compatible license as the original.You may do so in any reasonable manner, but not in any way that suggests the licensor endorses you or your use. attribution – You must give appropriate credit, provide a link to the license, and indicate if changes were made.to share – to copy, distribute and transmit the work.Joel Laurencio II Aptana Studio 3 TVET - 211 JRevision 0. This file is licensed under the Creative Commons Attribution-Share Alike 3.0 Unported license. o Right click the file o Select Open with o And select Web browser Web browser (firefox) Using Aptana Studio Web Browser of 18 Introduction to BSIT - 2A Date developed: Issued by: Mr. ![]() GFDL GNU Free Documentation License true true A copy of the license is included in the section entitled GNU Free Documentation License. Permission is granted to copy, distribute and/or modify this document under the terms of the GNU Free Documentation License, Version 1.2 or any later version published by the Free Software Foundation with no Invariant Sections, no Front-Cover Texts, and no Back-Cover Texts. Note: if the screenshot shows any work that is not a direct result of the program code itself, such as a text or graphics that are not part of the program, the license for that work must be indicated separately. See version 2 and version 3 of the GNU General Public License for more details. This work is distributed in the hope that it will be useful, but without any warranty without even the implied warranty of merchantability or fitness for a particular purpose. There then was a file called AptanaStudio3.exe which starts Aptana Studio, as expected. This work is free software you can redistribute it and/or modify it under the terms of the GNU General Public License as published by the Free Software Foundation either version 2 of the License, or any later version. On my Windows 7 64 bit machine, I found it correspondingly in C:\UsersUSER\AppData\Roaming\Appcelerator\Aptana Studio. You may use it freely according to its particular license. This screenshot either does not contain copyright-eligible parts or visuals of copyrighted software, or the author has released it under a free license (which should be indicated beneath this notice), and as such follows the licensing guidelines for screenshots of Wikimedia Commons. The manufacturer warrants the powder-coated, baked-on finish for six years from the date of installation. They’re suitable for 5”, 6”, or 7” gutters. K-Screen Gutter Guards are available in flat and ridged versions and come in four-foot lengths. Either option is a quick and easy installation by gutter professionals like those at Energy Pro. They fasten to the gutter either by snapping into the gutter itself or by snapping up under the roof shingles. No screws or fasteners are needed to install K-Screen Gutter Guards. These expanded metal gutter guards have a baked-on powder coat finish which reduces corrosion. ![]() This guard deflects debris while allowing rainwater to flow easily through the perforations and into the gutter. K-Screen Gutter Guard keeps debris out of the gutters by using a perforated guard. Whether your home is in Overland Park, Prairie Village, Olathe, or Leawood, Kansas or Liberty, Lee’s Summit, or Kansas City, Missouri, Energy Pro has a gutter guard solution that works for your home, protecting your investment – and your family – for years to come.Įnergy Pro is pleased to offer Kansas City homeowners three gutter guard solutions from Lansing Gutter Systems. We offer top-selling, high-quality gutter guard systems for homes in this region. Keeping your gutters clean is as important as having that gutter system in the first place.Įnergy Pro Windows and Siding is your go-to gutter guard specialist in the Kansas City area. However, your gutters can’t do the job they were designed for if they’re full of leaves, branches, and other debris. Your gutter system channels water off the roof into the downspouts and carries it away from your home’s foundation. Good-quality gutter guards not only make gutter maintenance virtually non-existent – eliminating the need to climb a ladder several times a year – but they also protect your investment. At that point, you’re looking at an expensive repair bill.Ī good gutter system, complete with gutter guards, can help prevent this situation. ![]() Strong winds, hail, and rain wreak havoc on your home over time, often causing damage that you don’t find until years later when it’s had time to deteriorate to where it’s obvious. Making cleaning a breeze and done within a fraction of the time.Your home is one of your most significant investments and should be protected as well as possible against damage from the elements. These Leaf guards help prevent leaves from clogging up your draining as well as making it easier to clean since the leaves sit on a shelf above your gutters drain. Gutter Brothers can create a custom leaf guard in Reno & Sparks and install it for you in your residential or commercial building. Adding that little touch of awesome to your home or business’s gutter solution. The chain spout is a new effective piece of art for your house. We can go with a traditional one or we can view other options like chain spout. Just like our custom gutter solutions we also create custom downspouts. Please call us for a quote or if you are looking for more information on the process and how you can get your Copper Gutters for your building!Īre you looking for something a little bit more unique in your gutters? Something that looks amazing and is functional as well? Check out our custom downspouts and rain chains. We specialize in creating custom Copper Gutters and installing them on your commercial or residential house. They can add a modern or rustic look to your house and will be turning heads of all your neighbors. ![]() Allowing water to be caught and brought elsewhere except copper gutters look amazing. Please feel free to call us to ask any questions you may have!Ĭopper Gutters give you the same functionality as a traditional gutter. Click here to view our Gallery! We can create a custom gutter plan for your house or commercial residence. ![]() Looking for some new gutters that don’t look like a million pieces? Seamless Gutter are the answer! What are Seamless Gutters? Instead of using many different pieces and attaching them with ugly connectors on your house we can create a custom single piece of aluminum that stretches from edge to edge. ![]() Remember to use the correct form of the adjective depending on your gender ( loco if you’re a male or loca if you’re a female)! What To Say When You’re EngagedĪfter you’ve been together for a while, the time may come when you and your partner decide to take your relationship to the next level and get engaged. This definitely isn’t a phrase you want to use on your first few dates, but it’s a good way to express how strongly you feel about someone after you’ve been together for a while. You can say this when you want to tell your partner that you’re crazy about them. It’s a somewhat silly way to think about love, but it works perfectly in this lighthearted expression. Supposedly, this sentiment derives its meaning from the fact that no two oranges are the same, so each half orange only has one possible match. Similar to English, this phrase is usually reserved for your significant other once you’re in a more serious relationship. This phrase, which literally means “you’re my half orange” in English, is a cute and humorous way to tell someone that they’re your other half. It’s a good sentence to use on special occasions though, like an anniversary. It’s also more of a dramatic phrase, so it’s probably not something you’ll say every day. This phrase should be reserved only for your partner because its meaning is more focused on expressing a romantic love. Te amo, on the other hand, is more a declaration of love. You should only say this to people you truly care about, and it’s a nice way to let them know how much you care. You can say this to friends and family, as well as your loved one. When you say te quiero to someone, it’s a more casual way to say you love them. In general, both of these phrases are used to say “I love you” in Spanish, but there are some slight differences that are worth knowing. Te quiero translates to “I want you” and te amo translates to “I love you”. Give these phrases a try when you want to convey your feelings. Once you’ve gotten past the first few dates and you’re in a steady relationship, it’s safe to use more romantic language when speaking with your partner. Everybody likes to hear that they look nice, and using this on a date can help keep a relationship moving in the right direction. It doesn’t come on too strong, but it definitely makes it clear that you’re attracted to the other person. This phrase is a good compliment for a date early in the relationship. Remember, Spanish adjectives have a gender, so it’s important to use the correct one when speaking to someone you like! You’ll want to be sure and say hermosa when talking to a female and hermoso when talking to a male. Literally “You look beautiful/handsome tonight”. It’s a nice, casual phrase that can encourage more conversation on a night out, and can help lead to another date! Te ves hermosa/hermoso esta noche. Using this phrase is a great way to let the other person know you enjoy spending time with them as a little more than a friend. It’s also a little more romantic than me caes bien, so it’s definitely appropriate to say on a second or third date. While the literal translation may sound a little strange in English, this is a completely acceptable phrase in Spanish. This means “I like you”, or literally, “you please me”. ![]() Using this phrase can pave the way to using more romantic phrases if the feeling is mutual. It’s a great expression to use if you want to test the waters when you’re talking to another person. Its direct translation in English, “you fall me well”, doesn’t make much sense, but this is a phrase you’ll hear in a lot of Spanish conversations. ![]() This is an innocent way to say “I like you” to friends and other people you enjoy spending time with. Here are some sentences you can use to keep the momentum going at the first stage of a relationship. There are a few phrases that can make it clear that you’re interested in the other person without being too forward. When you’re first getting to know someone, it’s best to play it safe. What To Say When You’re Getting To Know Someone These are 20 ways to express love in Spanish, based on the different stages of a relationship. Don’t worry though! Clozemaster is here to help. While there are a lot of resources to learn Spanish, not all of them cover romantic expressions. You don’t want to accidentally confess your undying love on a first date, and you definitely don’t want to tell your spouse you love them like a sibling either. If you find yourself in a relationship with a hispanohablante (Spanish speaker) someday, it’s important that you know the best ways to say how you feel in Spanish. Because it is one of the most widely-spoken languages in the world, there are a lot of unique ways to say “I love you” in Spanish. Using the right words to convey your feelings in another language can be really difficult, and Spanish is no different. It’s likely that you didn’t set up the gates in your automated compensation wizard correctly (see more on that here), or you tried to do manual compensation and made a mistake. Being a relatively new CyTOF user, I recently conjugated and titrated almost all my antibodies on helios. The easiest explanation for this situation is that you just didn’t calculate compensation correctly, so you need to go back and fix it. The GatingStrategy class supports the linear, log, channel ratio, inverse hyperbolic sine (asinh), hyperlog, logicle and FlowJo biexponential transformations, as well as Boolean, quadrant, rectangular, elliptical, and polygon gates. So my question: how should I deal with this spillover? Specifically, how do you generate a proper compensation matrix for CyTOF data? I've tried to explain it with some figures (see the PDF in the link below, sorry I didn't figure out how to easily insert pictures into this post) In order to determine the concentration of antibody to use, I have been looking at the separation of signal (low vs high) and the amount of spillover the antibodies generate.Ĭoncerning this last point, I'm not really convinced that compensation is not required for CyTOF. Data were analysed using FlowJo v9 (Tree Star, Inc.) and DIVA softwares (BD Biosciences). Compensation matrices can be read in from multiple sources or created de novo from a set of compensation bead files. I did this to the best of my abilities, but because I'm generating a panel to study mammary tissue, I don't have (m)any ab's that are exclusive.Īll comments and help are much appreciated! I asked around about this and (if people even bother with spillover) they tell me to properly titrate and swap metals around so you don't end up with weak ab's next to strong ab's and also you should know which ab's are mutually exclusive (so you know a certain cell can never have both antibody A and antibody B on it and if you do see it, you gate it out). I prepare my singles similar way as my samples using the same vile of antibody and same staining time, the only difference. I've tried applying compensation matrices to CyTOF data to deal with spillover, but for reasons I don't entirely understand it never seems to work particularly well. I use the CytoFLEX-S machine to run my samples and use FlowJo to analyze. In the case of isotopic impurity in particular, which should be fixed, I would imagine that you should be able to apply a standard matrix based on the known isotopic ratios, but when I've tried to do this the resulting increase in negative noise seems to outweigh any benefits in reducing spillover (much like what you experienced). The compensation matrix is represented by the grid to the left of the sample name. I believe the issue is related to the fact that even if the spillover in channel B may be a fixed percentage of channel A, at lower values of A it falls below the limit of detection in B. Create new, apply old, or compare different matrices. ![]() This means that instead of increasing linearly as signal A goes up, there is a lower/intermediate range of channel A where these is no apparent spillover into channel B and then an uptick once you hit the limit of detection (sort of a "hockey stick"). |
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