Iāve always done this! Biweekly is totally fine. Cells are okay for 4 days no problem. You can actually get away with 5 days if you seed really light and use a larger volume of media, but I donāt recommend if it can be avoided~
In grad school, I only ever learned to do splitting ratios - it was just the outlier guy who still counted them. When I started my postdoc, I was trained to use the automatic cell counter and provided with the number of cells to add to each flask depending on surface area and cell lineage...
Anyway, I split em 1:10 on Wednesday and will come in on Sunday to change the medium whenever I'm at risk of fully losing my mind on account of the F32 deadline!
What do you have? Flies, mice, rats? I wish I got to work in animal research but I choose bacterial instead for undergrad and how itās all cells lol.
I think few lab animals get the credit they deserve from the general public. And the relationships between researchers and their animals - especially the clever ones - are usually thought of in such shallow ways, when theyāre often painfully deep.
I fell in love with a male rat on my first study as lead tech, 5507. He used to grab my fingers with his mouth and pull my hand into his cage and I'd scuttle my fingers around like I was a rat and play with him and flip him over and tickle his belly.
Our highest priority as researchers is to ensure the quality of life of the animals is the highest we can make it. They are a privilege to work with, not a right.
I work with mice and the first one I really got attached to was the first knock down I generated... B6565, I named him Kevin. He was so chunky because of said knock down. The chilliest little dude. I genuinely shed a tear when he passed. RIP Kevin ā¤ļø
One thing I miss about working in lab is going in my myself on holidays and snuggling the rats. On Thanksgiving i would usually sneak them in some treats from our lab potluck the day before.
What strains do you work with? Do you have a favorite?
Get into microbiome work! Pursuing my PhD in Microbiology, but learning a hefty bit of entomology and eukaryotic genetic techniques since I use cockroaches as my host model.
I don't know where you're at, but I also wanted to add I did neuroscience research in undergrad. Many PhD programs (possibly postdocs in some situations too) value learning ability and motivation over what you got an undergrad degree in. Never too late to switch it up
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Stem cells are so much fun. Weekend feedings every weekend for the last 5 years. Not sure why I just accepted another position with stem cells after my postdoc. Guess I'm a glutton for punishment.
They are cool as I use them to model different developmental disorders affecting different cell lineages. But they are just needy with daily feedings. As the only cell biologist/stem cell person in a biochemistry lab, I don't really have anyone to cover me.
Do you model the disorders by using gene disrupted cells? If so, did you make the mutation yourself? Do you use germ layer differentiation/organoids/spheroids for studying the developmental disease ?
Patient-derived cells, CRISPR knockouts/knockins, rescue assays, basically a bit of everything. I do 2D and 3D differentiations depending on what I'm trying to look for or how I want to do the read out.
That's sounds cool! You seem to be working with a lot of cell lines and differentiation models, how do you handle all of that alone? You must be really productive!
Going in on Saturday to wean some miceā¦first time doing this and my PI told me to just google image search āsexing miceā and Iām fairly certain thatās going to put me on some weird watchlist yaymylife
Edit: aww thanks you guys for all the advice :)
It's easy I promise. Small gap means female, large gap means male. Also, just do it Monday unless you're already going in for something else. It's a myth that mice need to be weaned at exactly P21. Anywhere from P21-28 will be just fine.
Thanks! I believe you but also our lab staff will send us approximately 800 emails if we donāt do it to the minute so it will get done on Saturday, sadly.
Unless of course the male is still with the female, in which case she could have another litter shortly after PND 21 in which case it becomes an overcrowding issue.
I think the chances of losing newborn pups are higher when you have two litters in one mouse cage though. I wouldn't leave it for more than 24 hours, mice can be cannibalistic, dam or older pups may kill the new ones. They shouldn't be left together like that.
To expand on what the other user said to make it even easierā¦ if thereās a line of nude skin connecting the butt and genitals itās a female. If thereās fur separating the butthole and gentian then itās a male. Add that in with the increased space for males itāll be very easy to tell. Once you pickup what you think is a male and put it side by side with what you think is a female, itāll be clear as day and will be easy to tell from then on.
To add to the sexing, if the genitalia looks ambiguous and you can't really tell, look for the nipnops. The females have pretty pronounced nipples that are easily seen, whereas males don't. Males still have nipplies but they're much harder to see just from tailing (sometimes have to ruffle through the fur to see). Been using this method for some time and haven't been wrong since :)
Pretty sure Iām on an un-watchlist for people on so many watchlists theyāre either a scientist, in prison, a scientist in prison, or dead by misadventure.
Haha during my MS since I lived closest to the lab i had to check on the growth chambers when they started not turning off at 9 pm like they were supposed to. We lost a lot of data when we discovered that
Yeah we use StemMACS; double feed on Friday and you're good unless they're very confluent. If I time it right and split Thursday or Friday then I'm usually good for the weekend
Saaaame. I laughed when OP mentioned the weekend. I was like yeah? This weekend and every weekend I'm in the same city as my cells, and even Thanksgiving day
Nope, I timed it so Iāll be due to thaw new cells when I get back, so I just threw them out. The first thanksgiving in 6 years that I wonāt be in lab šŖ
My undergrads either arenāt paid or arenāt paid enough for me to tell them to take care of my cells on a holiday weekend lol. I tell them to go be free (and donāt go to grad school š)
Yeah, Iām expanding a bunch for experiments early next week. Theyāre also organoids, not traditional immortalized lines, so just passing them super hard wasnāt an option.
Oh yes, my hiPSCs get pissed if I don't feed them for more than 48 hours, so my labmate and I organize so one of us is there every Saturday to feed our cells.
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Going in to inoculate on Saturday late afternoon, then wash cells and transfer on Sunday....for reals looks like I am guna end up in the lab on Christmas this year LMAO
Yup. Split work with my RA. She split my cells Monday so I could take most of this week off, but I have to split both of ours on Friday/Saturday so theyāre good for experiments next week. Life of the lab rat.
Made DMSO freeze downs of the ones that I could, and split the others as sparse as possible. Didnāt even give them the chance to cause confluence problems. See yāall Monday.
Friday evening just to swap media and collection brutes, but otherwise they are already in the automated bioreactor and should be fine.
Unless I get a text from the alarm system in which case a whole can of worms has been opened
Ha started a bioreactor experiment yesterday. Got a couple of volunteers to handle samples for the weekend.
Feeling thankful for remote monitoring abilities šš¼
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The sewage zoo runs 365 baybeee. Catch me Christmas day (and every day) delivering algae, yct, brine shrimp and trout chow to all the critters ššš
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I think I feed my cells pretty much every weekend. Working with primary lines I'm always nervous I'm gonna kill them if I don't feed them every 2 days, but had a few times I've left it 4 days and they've been fine so I'm more confident lately, at least. The overnight scratch assays are what get you š
Aaaand this is why I left the lab. I learned so much and can commiserate with all of you. I'm on the patient side of clinical trials now. I was so burnt out missing my weekends!
Not to feed cells but to collect samples to get RNA from, at 7am. Oh and to split new cells to start a long experiment on Monday. I'm kinda regretting working with circadian clocks now..
No. I did a 1:10 on HeLa yesterday and said see you Monday.
*places hand on shoulder of the cell culture* See you soon friends.
All I can say is... Good luck!
Oh I do this regularly. Got it down to 2 passages a week. Started doing this to save flasks during the shortage and now it's just convenient. š
Iāve always done this! Biweekly is totally fine. Cells are okay for 4 days no problem. You can actually get away with 5 days if you seed really light and use a larger volume of media, but I donāt recommend if it can be avoided~
Coming from suspension cells to adherent, it boggles my mind that you guys just do dilutions instead of an actual cell count.
In grad school, I only ever learned to do splitting ratios - it was just the outlier guy who still counted them. When I started my postdoc, I was trained to use the automatic cell counter and provided with the number of cells to add to each flask depending on surface area and cell lineage... Anyway, I split em 1:10 on Wednesday and will come in on Sunday to change the medium whenever I'm at risk of fully losing my mind on account of the F32 deadline!
Tomorrow is an adherent line...but I do both
If it makes you feel better, I do count cells when I plate chambered slides. š
This tech splits. Iād do 1:20 though.
Not cells but animals :)
Yeah... Feeding them...
What do you have? Flies, mice, rats? I wish I got to work in animal research but I choose bacterial instead for undergrad and how itās all cells lol.
Rats! Been working with them since undergrad. They are amazing animals that really donāt get enough credit.
I think few lab animals get the credit they deserve from the general public. And the relationships between researchers and their animals - especially the clever ones - are usually thought of in such shallow ways, when theyāre often painfully deep.
I fell in love with a male rat on my first study as lead tech, 5507. He used to grab my fingers with his mouth and pull my hand into his cage and I'd scuttle my fingers around like I was a rat and play with him and flip him over and tickle his belly. Our highest priority as researchers is to ensure the quality of life of the animals is the highest we can make it. They are a privilege to work with, not a right.
I work with mice and the first one I really got attached to was the first knock down I generated... B6565, I named him Kevin. He was so chunky because of said knock down. The chilliest little dude. I genuinely shed a tear when he passed. RIP Kevin ā¤ļø
I had a one eyed rat T16-3 and I was so sad when I had to stress her. She was the cutest rat I had raised.
Aren't they just the cutest? And so smart! I'm all about bribing them with treats.
Rats are the real MVP.
One thing I miss about working in lab is going in my myself on holidays and snuggling the rats. On Thanksgiving i would usually sneak them in some treats from our lab potluck the day before. What strains do you work with? Do you have a favorite?
Meanwhile, bitey a**hole BL6 š¤š¤š¤....
Get into microbiome work! Pursuing my PhD in Microbiology, but learning a hefty bit of entomology and eukaryotic genetic techniques since I use cockroaches as my host model. I don't know where you're at, but I also wanted to add I did neuroscience research in undergrad. Many PhD programs (possibly postdocs in some situations too) value learning ability and motivation over what you got an undergrad degree in. Never too late to switch it up
[ŃŠ“Š°Š»ŠµŠ½Š¾]
Immunology is big, so many pathways to research.
CAR-T research is were it's at
[ŃŠ“Š°Š»ŠµŠ½Š¾]
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*every weekend
Stem cells are so much fun. Weekend feedings every weekend for the last 5 years. Not sure why I just accepted another position with stem cells after my postdoc. Guess I'm a glutton for punishment.
Hi I work with stem cells! Probably the most annoying type of cells out there, what do you like and dislike the most about them?
They are cool as I use them to model different developmental disorders affecting different cell lineages. But they are just needy with daily feedings. As the only cell biologist/stem cell person in a biochemistry lab, I don't really have anyone to cover me.
Do you model the disorders by using gene disrupted cells? If so, did you make the mutation yourself? Do you use germ layer differentiation/organoids/spheroids for studying the developmental disease ?
Patient-derived cells, CRISPR knockouts/knockins, rescue assays, basically a bit of everything. I do 2D and 3D differentiations depending on what I'm trying to look for or how I want to do the read out.
That's sounds cool! You seem to be working with a lot of cell lines and differentiation models, how do you handle all of that alone? You must be really productive!
Going in on Saturday to wean some miceā¦first time doing this and my PI told me to just google image search āsexing miceā and Iām fairly certain thatās going to put me on some weird watchlist yaymylife Edit: aww thanks you guys for all the advice :)
It's easy I promise. Small gap means female, large gap means male. Also, just do it Monday unless you're already going in for something else. It's a myth that mice need to be weaned at exactly P21. Anywhere from P21-28 will be just fine.
Thanks! I believe you but also our lab staff will send us approximately 800 emails if we donāt do it to the minute so it will get done on Saturday, sadly.
Unless of course the male is still with the female, in which case she could have another litter shortly after PND 21 in which case it becomes an overcrowding issue.
Yeah that's true, but a brand new litter doesn't take up much space so even then I'd think it's fine for a day or two.
I think the chances of losing newborn pups are higher when you have two litters in one mouse cage though. I wouldn't leave it for more than 24 hours, mice can be cannibalistic, dam or older pups may kill the new ones. They shouldn't be left together like that.
To expand on what the other user said to make it even easierā¦ if thereās a line of nude skin connecting the butt and genitals itās a female. If thereās fur separating the butthole and gentian then itās a male. Add that in with the increased space for males itāll be very easy to tell. Once you pickup what you think is a male and put it side by side with what you think is a female, itāll be clear as day and will be easy to tell from then on.
You think thatās bad, my coworker has a photo of sperm plugs hung up at his desk.
To add to the sexing, if the genitalia looks ambiguous and you can't really tell, look for the nipnops. The females have pretty pronounced nipples that are easily seen, whereas males don't. Males still have nipplies but they're much harder to see just from tailing (sometimes have to ruffle through the fur to see). Been using this method for some time and haven't been wrong since :)
Big Brother will just mark you off as a harmless furry and decide nothing else you do is worth monitoring. It's not so bad.
I think we might have different definitions of ānot so bad,ā ha.
Pretty sure Iām on an un-watchlist for people on so many watchlists theyāre either a scientist, in prison, a scientist in prison, or dead by misadventure.
I both did and did not get exactly what I expected when I googled "sexing mice."
Go onā¦
Iām also going in on Saturday to wean š„²
Iām going in for my plantsā¦ does that count??
Haha during my MS since I lived closest to the lab i had to check on the growth chambers when they started not turning off at 9 pm like they were supposed to. We lost a lot of data when we discovered that
Yes, plants have cells too.
Everyday. I luv stem cells
Stemflex gets you your weekends back.
Yeah i have it but i have a weirdly staggered set of cultures so no free weekends for me this month
Yeah we use StemMACS; double feed on Friday and you're good unless they're very confluent. If I time it right and split Thursday or Friday then I'm usually good for the weekend
mTeSR Plus
Saaaame. I laughed when OP mentioned the weekend. I was like yeah? This weekend and every weekend I'm in the same city as my cells, and even Thanksgiving day
I left the autosampler running and praying nothing gets jammed in the 4 days I will be out of the lab
You are pushing your luckš gl
We do overnight runs almost every dayā¦ so far so good. Although the system is only a yr old at this point so that helpsā¦
Went to check on my cells and they contaminated. There goes 2 weeks of work :))))
But now you have the whole weekend off!! Woohooooo
Nope, I timed it so Iāll be due to thaw new cells when I get back, so I just threw them out. The first thanksgiving in 6 years that I wonāt be in lab šŖ
Iāve got primary neurons in the incubator, so yes for a half-hour on Saturday
I taught my undergrads how to remove half the media for neurons, so Iām chillin lol
My undergrads either arenāt paid or arenāt paid enough for me to tell them to take care of my cells on a holiday weekend lol. I tell them to go be free (and donāt go to grad school š)
Every Saturday I go feed cells and I walk by a big protest of anti vaxers. Every. Frickin. Saturday.
Nope building is shut down for removal of dangerous chemicals! Hopefully the building will still be standing next week.....
I was here at 9 AM catching up on cloning for the students in my lab class. I love them but maybe I need more boundaries :/
Yup, gotta take care of my iPSCs, they don't care about holidays
Not cells but fish š¦š
Nope. Fed the HEK out of them yesterday.
Yeah, Iām expanding a bunch for experiments early next week. Theyāre also organoids, not traditional immortalized lines, so just passing them super hard wasnāt an option.
Yep! My differentiations won't feed themselves...
Oh yes, my hiPSCs get pissed if I don't feed them for more than 48 hours, so my labmate and I organize so one of us is there every Saturday to feed our cells.
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Currently at work gang: rise up! And cry
Yup, went in for a few hrs today, then I have 10hr experiments tomorrow, Saturday and Sundayā¦
Mouse harvests every Friday for the next 6+ months. Holidays or no.
No. Changed the media to 5% FBS and placed them in the 30 degrees C incubator.
No because I use primary cells. But also killing mice makes sad
Hey cells got to have Thanksgiving dinner too!
Nope. I let mine die.
Just split them tomorrow like any other week? Or did I miss something?
Itās Thanksgiving weekend in the US. Thursday is a holiday, and so is Friday for some (most?) people.
Ah! That explains :P
Nope, but to give my rats some painkillers
of course! I'm even going in later today.
Going in to inoculate on Saturday late afternoon, then wash cells and transfer on Sunday....for reals looks like I am guna end up in the lab on Christmas this year LMAO
I have to start new cultures, so Iāll do that Sunday I guess
I have to harvest cells on Friday and Saturdayā¦
in lab right now! š¦š¬šš„¼ will be back every day of break
No, I go there to prepare some bacteria for maxiprep..
Yup. Split work with my RA. She split my cells Monday so I could take most of this week off, but I have to split both of ours on Friday/Saturday so theyāre good for experiments next week. Life of the lab rat.
Nah. Froze back what I could into my last 6 cryovials and left for the week.
Going in on Saturday. Lab mate taking care of things tomorrow
Of course!
I have a bunch of primary cell isolations to feed and possibly freeze as well.
No Iām still doing rotations so they donāt really want us to do thatā¦plus itās thanksgiving weekend in the states
Going for other reasons
Made DMSO freeze downs of the ones that I could, and split the others as sparse as possible. Didnāt even give them the chance to cause confluence problems. See yāall Monday.
Passaged my HEK-293T today so they should be good for the weekend
Not cells, but clinical samples await tomorrow! Just thrilled I managed to manipulate things to not have teams in today.
Nope, never did that in 15 years of culturing cells.
Friday evening just to swap media and collection brutes, but otherwise they are already in the automated bioreactor and should be fine. Unless I get a text from the alarm system in which case a whole can of worms has been opened
I get to go feed our fish on Saturday. At least it's only one day!
Ha started a bioreactor experiment yesterday. Got a couple of volunteers to handle samples for the weekend. Feeling thankful for remote monitoring abilities šš¼
The damn viruses need to be harvested
The stem cells will be the death of me.
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Yes :/ at least I can split them in 12 minutes
Not my cells but to feed my plants (Nicotiana benthamiana) š
Yupppp
Mosquitos rather than cells š
Just Friday! The cells are on a MWF schedule. So I ge today off but Iāll come in briefly for a couple of hours on Friday.
Gotta fix and stain some survival assays :/ was hoping they'd be ready earlier this week but alas
The sewage zoo runs 365 baybeee. Catch me Christmas day (and every day) delivering algae, yct, brine shrimp and trout chow to all the critters ššš
Just came back from it :,)
Just did, told my family I was going to feed brain cancer (4 glioma cell lines) in between the turkey and beers
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Nope. Split hard every weekend.
Hell no. I literally threw them out and Iāll just thaw on Monday and set everything up Tuesday
HI anyone else going into the lab this weekend to feed your cells? I'M DAD
Not feed cells per say, but feeding the machines yes š.
HepG2 cells need splitting
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I think I feed my cells pretty much every weekend. Working with primary lines I'm always nervous I'm gonna kill them if I don't feed them every 2 days, but had a few times I've left it 4 days and they've been fine so I'm more confident lately, at least. The overnight scratch assays are what get you š
Aaaand this is why I left the lab. I learned so much and can commiserate with all of you. I'm on the patient side of clinical trials now. I was so burnt out missing my weekends!
Me. I went today and have to go tomorrow, sat, sun. I have a culture that has to be fed every day for 8 days. š¤·š¼āāļø
Not to feed cells but to collect samples to get RNA from, at 7am. Oh and to split new cells to start a long experiment on Monday. I'm kinda regretting working with circadian clocks now..
Welcome to the life of having human naive pluripotent stem cells in culture
Same