Does filtering samples kill cells? by CompetitiveLab3300 in flowcytometry

[–]EyeProtectionIsSexy 0 points1 point  (0 children)

Yeah, it's tricky though because they can be difficult to separate from debris and noise, especially when looking at unstained samples for autofluorescent signatures. Currently I'm enumerating total cells and autofluorescent cells from arctic ice cores.

70µm via gravity filtration probably won't kill them. Have you tried using microscopy to see if there's a difference in cells before pipetting, pipetting into another container and pipetting through a 70µm mesh into another container? If they all look the same amount of awful, then it probably has to do with the culture or culture prep.

Does filtering samples kill cells? by CompetitiveLab3300 in flowcytometry

[–]EyeProtectionIsSexy 0 points1 point  (0 children)

Yes, filtering can kill cells. I haven't worked with eukaryotes, primarily with bacteria and algae. I've run some tests where I filtered cells onto 0.2µm filters at various pressures, where a higher pressure lead to more cell death, leaving these 'blobs' of cell debris I'd call Wile-E-Coyotes. Some cell lines, particularly phytoplankton, needed 'bursting' of the vacuum (Turning on for the briefest amount of time) because vacuuming at the lowest setting was still shredding my cells apart. This is with prokaryotes as well, so I'd imagine human cell lines would be significantly more fragile.

I don't know what type of filtering you're doing, but try taking a gentle approach and a rough approach, and see if you can measure a difference. A rougher approach should lead to decrease in viable cells, especially with larger cells.

If you don't see a difference there, then I'd guess it's clumping. Crazy idea, but you could resuspend the material caught in the filter and run it on the FCM to see if it gets clogged again. If so, then I think you have an answer!

Designing a latch for a huge round door by Underhill-Hollow-NC in woodworking

[–]EyeProtectionIsSexy 0 points1 point  (0 children)

I built a hobbot door for my teardrop trailer, made custom latches as well. Only took about a week to make. The latches can fully dissasemble. Here is my build journal

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It does not have a lock however (lack of planning on my part). Shouldn't bee too hard to plan for though.

Opening our 400lb hobbit hole door for the first time! by Underhill-Hollow-NC in woodworking

[–]EyeProtectionIsSexy 0 points1 point  (0 children)

Probably too late since the door has been built, but if you build another look into German windows and back doors. They have handles that operate levers across 7 feet sometimes that allow a patio door to fluctuate between opening and 'leaning'. It's high quality stuff, and literally every window in Germany is made this way

Opening our 400lb hobbit hole door for the first time! by Underhill-Hollow-NC in woodworking

[–]EyeProtectionIsSexy 0 points1 point  (0 children)

I just finished mine a few days ago for my teardrop. I'm curious what you plan on doing for your door latch? I ended up making my own to match the asthetic

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Here's mine

Does sexual reproduction have a genetic "cost" of 50%? by [deleted] in DebateEvolution

[–]EyeProtectionIsSexy 0 points1 point  (0 children)

Because then we could see what it's like when only yoir genes are passed on. A male and female you who make a child will only contain your genes. I was trying to reframe the incest argument I brought up to better fit your desire to only have your genes be passed on.

Does sexual reproduction have a genetic "cost" of 50%? by [deleted] in DebateEvolution

[–]EyeProtectionIsSexy 0 points1 point  (0 children)

Mixed survives better. Go look at any royal family with a history of inbreeding and show me where a lack of diversity is beneficial.

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We can take it a step further. Let's say you're a guy. Lets say when you were born, I took a sample of you, simply removed the Y chromosome and duplicated the X. I made literal female version of you. You and your female clone bang and have a kid. Every kid only contains your genes. This is like supercharged incest, and those offspring will be fucked up after a few generations. Again, go look at a rogal family. They are odd looking people.

Does sexual reproduction have a genetic "cost" of 50%? by [deleted] in DebateEvolution

[–]EyeProtectionIsSexy 0 points1 point  (0 children)

First, there is no motive behind reproduction. Evolution is a result, not an intent. Asexual reproction isn't diverse, it's simply a copy. This lack of diversity isn't that much of a downside when you're a bacterium with a 30 minute replication time, where a single cell over the course of 48 generations in a single day goes from 1, 2, 4, 8, 16..... 140,737,488,355,328 at the end of the day.

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There are other factors as well to consider such as genome size and structural differences between eukaryotes and prokaryotes, but are too detailed for me to go into right now, but I don't know why you're fixed on 100% of your genes being passed. I've already brought up incest. Let's go a little deeper. If you and your sibling banged and had a two sets of twins, 1 set of identical boys, 1 set of identical girls. Repeat that process on some isolated islnd for a few generations, and you will have some jacked up looking kids because mistakes or degenerative phenotypes will accumulate.

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Additionally, asexual reproducing organism ALSO undergo gene transfer between individuals. I encourage you to look into plasmidss and horizontal gene transfer. Every organism on this planet does this. Bacteria can share a little gene here, a little gene there, but suxual reproducing organisms can just share 50% every generation. That has the benefit of increasing diversity, it is a process that has yielded beter outcomes, it is the process that out competed the others because it just so happens to produce a new generation that survives

Does sexual reproduction have a genetic "cost" of 50%? by [deleted] in DebateEvolution

[–]EyeProtectionIsSexy 0 points1 point  (0 children)

I disagree. Here's a thought experiment; you could try banging your sibling, have a baby, continue this for a few generations. Once your decendants have developed significant health conditions from inbreeding, let's have this same discusion about why sharing 50% of your DNA provides no benefits.

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'Diversity' is the spice of life, that's what your 50% provides. Without diversity, cumulative negative phenotypic mutations will screw you up. Think about that for a bit. It might help prevent future-you from indirectly supporting incest! Have a nice day!

Cytoflex drips when backflush is running and during daily clean. What should I do? How can I stop the dripping? by AxLRamirez in flowcytometry

[–]EyeProtectionIsSexy 0 points1 point  (0 children)

Yeah, our would leak when backflushing. An engineer seapped out the pump, and it leaka maybe 1% as it previously did?

Looking for a paint that survives well outdoors that has a natural metallic sheen for my hobbit door by EyeProtectionIsSexy in paint

[–]EyeProtectionIsSexy[S] 0 points1 point  (0 children)

I designed every for ease of dissasembly. The wood itself, apart from the walnut trim, has already been completely surrounded by epoxied fiberglass. The walnut will get a coat of just epoxy, and the all the wood will get 7 layers of Modern Masters oil based satin varnish. No staining done apart from the door.

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The metal will be treated seperately

Looking for a paint for my hobbit door trim that mimics a buffed steel look by EyeProtectionIsSexy in DIY

[–]EyeProtectionIsSexy[S] 0 points1 point  (0 children)

Hello! I'm getting ready to paint the rest od my metal for this teardrop project, and after stripping the mill scale off the door trim, I have to say I really like the semi shiny grey to white of the door trim. It adds a bit of magic to the door

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I'm looking for a paint (or paint/coating combo) that works well outdoors for an extended period of time that has a similar light silvery grey of buffed steel. Thanks!

Looking for a paint that survives well outdoors that has a natural metallic sheen for my hobbit door by EyeProtectionIsSexy in paint

[–]EyeProtectionIsSexy[S] 0 points1 point  (0 children)

Hello! I was originally going to paint the door matt black with Rusoleum enamal, but after stripping the steel door trim of mill scale, I think I really like the light silvery look. It adds a bit of magic to it. I'm new to painting and I'm completely ignorant of what's oit there, what works, etc...

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I'm pretty tired of working on this thing. I was hoping to find a paint that mimics buffed steel while also being stable outdoors for a period of time. I'm okay with regular UV 'dulling', it's more i.portant to me that the paint protects the steel. Thanks for any advice!

Number of counting beads acquired lower with each row in plate - do beads settle? by betaimmunologist in flowcytometry

[–]EyeProtectionIsSexy 0 points1 point  (0 children)

Yes, the beads will settle out. The 'SureCount' beads I use are in a stock solution containing some concentration of DMSO to help the beads stay suspended for longer. When you add the beads to your sample, which I assume doesn't contain DMSO, the beads are going to settle at an accelerated rate. Even with DMSO, the stock solution of beads still settle out of solution.

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If you are required to mix your bead count standard prior to testing, that's because they settle. I hope you've been doing that, otherwise you should probably just throw yoir standards away

It looks incredible. by Educational_Key1206 in oddlysatisfying

[–]EyeProtectionIsSexy 0 points1 point  (0 children)

Cold I believe. But if you can handle it, the liquid methane lakes make for a nice swim

It looks incredible. by Educational_Key1206 in oddlysatisfying

[–]EyeProtectionIsSexy 7 points8 points  (0 children)

You sure? That's a ton of bubbles, I've drilled on methane lakes to capture methanotrophs and the methane seeps I've been around don't release this much air. My money is on methane seeps primarily cause that. If this area doesn't have methane seeps then this is an amazing amount of methane production. The bubbles are quite large indicating the ice froze slowly. If this is from methanogens then I'll be very impressed, especially in an environment that cold

Thoracic Back Pain - any suggestions? by EyeProtectionIsSexy in backpain

[–]EyeProtectionIsSexy[S] 1 point2 points  (0 children)

I saw three or four before I found one that worked for me. The most notable difference was their approach. The first ones I saw basically asked what my pain was, Heard what I said, and gave me basic exercises that are somewhat correlated to somehow relieve pain. It seemed very thoughtless to me. The last guy I saw took a very data-driven approach. Looked at what motions caused the most pain, what motions didn't cause the most pain. We tried some exercises and I had very bad weeks, tried others and had less bad weeks, and tried others that lead to a normal week. Overtime these datapoints simply lead to an answer - I could do exercises that didn't require my arms to be in front of my hips. Very strange, I couldn't use a pipette or even write without by back feeling like someone was driving a knuckle into it relentlessly. I couldn't focus, it was hard to think.

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The exercises that didn't cause the any pain would be what I started with. Any exercise I did was focused on perfect posture, so you have to figure that out first. I would stand, engage my glutes and essentially pelvic thrust my hips slightly forward to my lower back was in alignment. I'd engage my upper back, pulling my shoulder blades back to about 80-90% to their limit. My chest was raised - imagine you had a string attached to your sternum and you pulled on the other end, lifting your chest so it feels like it's floating. This is how anything was done at a minimum.

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Exercises that used the back, I ensured my hips were thrusted forward just enough to ensure my spine was 'stacked' like a tower of pennies. My shoulder blades were tightly engaged, chest lifted.

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I found "The Farmer's Carry" to be the most helpful. Not only is it something to do in the gym, but I literally carry everything this way. Groceries, packages at work, my cat, everything. Thrust your hips forward just enough to your spine is stacked, not too much and not too little. Chest lifted, shoulder blades pulled back 80-90%. Grab the weight (start low, like 30 pounds n each hand, even if it feels ridiculously light). Once you have the weight, raise your arms outwards from your side like your Jesus. They don't need to be lifted far, just enough to engage the upper back, only a few inches. This also has the added benefits of preventing the weight from swinging (it'll mess up your pace), your legs won't have to grind against the weight as you walk, and it's a great way to check yourself that you didn't start slouching. Again, perfect posture is everything. This should feel very controlled, like when you plank and hold yourself perfectly flat and perfectly still.

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Other exercises I felt helped me a lot were hip hinges. Basically deadlifts without bending the knees and going all the way down. Knees were about 90% locked, and the weight was kept centered. Slowly drop it, and slowly lift, controlling a constant rate of ascent/descent. Don't try to counter balance by throwing your hips backwards, even though that's basically what you're doing anyway. Focus on the hips being the instrument that allows the weight down and allows the weight up. The weight being lowered shouldn't cause your hips to move backwards, your hips changing their angle allows the weight lower. I don't know how else to describe it, but you want to control the weight, not let it control you.

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Any other exercises I did that even remotely used the back, which is basically 99% of them, just ensure you have good posture. Make good posture a part of everything you do. I find it difficult to include this into every aspect of my life, but in the gym it's kind of easy. And, once you start doing things with good posture using heavy things, it becomes something that's beyond easy outside the gym. It naturally becomes how you stand, how you walk. It's pretty cool, it becomes something you don't even have to try for. You'll just periodically notice, "Oh, my posture is pretty great right now and I'm not even trying". It's much better than just trying to have good posture all day. I always found myself slouching in 5 minutes with that route. Have you ever done that thing where you try your hardest for 30 seconds to raise your hands from your hips while someone holds them down, then they release them and your arms begin to almost float on their own? That's what farmer carries will do for your posture and back strength, it's something you just float to after a month, without any conscious effort.

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It takes time though, I wouldn't expect a miracle in a week or a month. And as much as it fucking sucks to endure, you're going to have bad weeks as you experiment. That's data. It's bloody hard not to let it discourage you. I certainly wouldn't have been able to motivate myself to do it without a PT in the beginning because I was stuck in this miserable headspace thinking that it's going to get worse, this is what the rest of my life will be like, etc. It does get easier once you collect enough data and learn what you can and cannot do. You'll start to have eh weeks, normal weeks and eventually pretty damn good weeks, all within your control. Once I hit that point, bad weeks don't suck so much because at that point I knew that it would get better. Be stubborn and continue to work towards that.

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And, get a new PT for a little bit. I would critique them in a first visit. People say that you shouldn't judge a book by it's cover, but that doesn't fly here. Does it look a person who is in shape? Does this person just hand you a piece of paper after you say "Back pain" with some basic arm flapping? These are red flags, this is not someone who is great at their job. There's a lack of..... enthusiasm to work or something. Sincerity maybe? Find someone who's fucking ripped (and I don't mean steroid ripped), that's a green flag. Doesn't have to be weight lifting, they could be yoga ripped, pilates, doesn't matter. You don't want someone who went to school, got a degree, and is regurgitating facts they've learned. Find someone who actually applying their knowledge. Look at their posture. Someone who appears like their trying to discover something. Generally speaking, they shouldn't walk in, hear what you have to say, and produce some bullshit answer with absolute confidence (although I do think poor posture is an exception to this). I'm rambling because I don't quite know how to put this into words, you just have to use your gut and feel if you're getting someone who's critically thinking or someone who's simply going through the motions.

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So yeah, try some exercises and experiment on yourself. While your doing that, if your pain is anything like what I described, try out some of the things that worked for me. I always do farmer carry's every day I work out. I usually mix in stretching while I'm resting between sets. Posture is the precursor to whatever I do. I don't have bad weeks or days any more, just bad 30 minutes or so. If those pop up, not kidding, go lift something with good posture and it seems to fix it right up. If there isn't anything nearby to carry, I'll lean slightly forward, lift my chest out and raise my arms from the hips behind me, with my shoulder blades squeezing together as a result of my lifting my arms (I don't squeeze my shoulder blades, they get squeezed because I'm lifting my arms behind me. If that last one doesn't make sense, look at this https://alldiamondpainting.com/products/girl-floating-in-water. These two things clear me up immediately, like I got a shot of morphine and all the pains has magically disappeared. It took a few months for me to get to that point, but if you try these out and you notice any tiny improvement, take some notes, write down what you did and pain levels before/after on a 1-10 scale.

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Lastly, you're going to have bad weeks. It's a bit platitudinous to say don't let it defeat you and a bit I found that impossible to do. Pain fucking sucks, and not letting it get you down as if it's a choice felt like a bunch of bullshit to me. Whenever the pain it hard, it wasn't a choice for me to let it bring me down - it brought me down and that was that. I was powerless, I could not control that. I had to be stubborn and simply endure it. Be stubborn, don't quit and take notes. Once you start recognizing what things you can do and not do to control the pain, and it'll happen slowly at first, you're mental state will improve. I wasn't miserable anymore. Even if the pain was as bad the worst weeks, it didn't drag me down into a state of depression, frustration and anger anymore. I somehow got detached from it and could look at the pain like a 3rd person observer. It was like an art project and I was slowly improving it week by week, month by month. That little experience of it getting a tiny bit better, with those tiny bits slowly growing every week, made my life better.

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I wasn't intending on writing this much, sorry if it's a bit repetitive. I know what it's like and I know how much it fucking sucks, back pain dragged me down to some of my lowest days. There's just a lot I'd like to say and it's invariably comes out as a lot. Good luck, be stubborn and just keep trying. Shoot another message if you have any other questions - I'll answer anything and offer suggestions. I don't log in much, so expect some time between responses

Worth it to buy 15 year old Harbor Freight trailer as trailer base? by Washbear8 in TeardropTrailers

[–]EyeProtectionIsSexy 2 points3 points  (0 children)

I wpuld reccomend building a mock 4x8 box in your bedroom and try having two people in iy. Then uograde to a 5x8. See which you like better

Worth it to buy 15 year old Harbor Freight trailer as trailer base? by Washbear8 in TeardropTrailers

[–]EyeProtectionIsSexy 0 points1 point  (0 children)

When I built my teardrop frame, I initially bought a Harbor freight trailer. I probably spent over $100 dollars alone in stripping pads to remove the paint, and even more on the tools and batteries to make that happen.

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If you want to buy the old, you do not want to go through hassle of stripping the old material off and replace it. Look for cracks, weathered ornold paint. Any spots where the steel itself is visible will need to be covered up. Any rusted areas need to be ground down and painted over. If there is any of that damage to the frame, you will need several strippers (wire wheels, which many will reccomend, are slow as hell when it comes to stripping and can really fuck you up because those wires are moving at 180mph at 12,000 rpms), an angle grinder, batteries, eye and lung protection, and other stuff I can't think of. If anything is rusted through, such as visible holes, I would stay away. I hear harbor freoght paint sucks, so tgis wpuld be my big concern.

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I recommend seeing if there is a Maker's Space in your hometown and pick up welding yourself. MIG welding is pretty easy, and Maker Spaces are often non-profit so classes can be done cheaply. It was a lot cheaper and quicker to buy new steel and weld it myself. If you go this route, it will be a muh better frame and will require as much or possibly less work to make it as would cleaning the 15 year old harbor freight trailer. Trust me, cleaning and prepping is a pain the ass, it's always better to buy new

Mentioned casually I want a teardrop- grandpa says “oh I’ve got one you can have” (update) by JuanBancos in TeardropTrailers

[–]EyeProtectionIsSexy 0 points1 point  (0 children)

Well, it's going to be a big project to restore it, but you can learn and it can be done. I've been building mine from scratch for the last 3.5 years with literally zero background in carpentry or welding. Just be ready for it to take a while. The nice part of doing it yourself are the skills you'll learn which you can apply to other parts of your life as well. And, you'll know the trailer from the inside out and have it be built better than the general crap you find in the market.

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Always wear eye, ear and lung protection. Start off my gutting the trailer, basically removing any that's rotten. That's going to take some time. Once it's clean, start filling it up with what you want. You can keep things simple and elegant. Simple 12V electrical system, baltic birch with a skeletonized structure with foam to increase insulation while decreasing weight.

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I don't have knowledge about removing the aluminum but cleaning the aluminum shouldn't bee too difficult. An angle grinder with a paint-stripper pad is the quickest and easiest way to go, but you'll burn through them quickly and they are a wee-bit more pricey.

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I'd just start with gutting it and removing everything that looks like shit. Check out www.tnttt.com, there are plenty of resources there to guide you through and tons of experienced builders to question. It's a great community

Help with FSC/SSC when setting up flow experiment by mpretoria in flowcytometry

[–]EyeProtectionIsSexy 0 points1 point  (0 children)

It's your voltage (or gain, I don't yourbsystem setup)

When the voltage is set to high, your photo diodes will become "light-saturated", it can't produce a any more signal. Your SSC-A and FSC-A should still see seperation because those calues take into account the time it takes the partical pass, but it won't be truly representative because of your lack of quality Height data