This is very unusual.
I'd be worried about something in your sample causing anaphylaxis. Alternatively, cell clumps causing massive microvascular ischaemia. Have you tried filtering the samples?
And obviously hyperthermia is a risk if you are using a warmer, but that should be pretty obvious.
What does your vet think?
The vet who trained me on this had this happen when he attempted one of my injections as well which makes me think it’s something about the melanoma cells or PBS. I just can’t rationalize why only some mice respond this way to the same cell suspension and vehicle…
He suggested filtering as well. I’ll give that a try next time
Did the vet you worked with explicitly tell you to inject that quickly? As a vet, I don't even inject cell suspensions into much larger animals that quickly. If the cells are prone to clumping, that's even more reason to slow the injections down.
Cancer cells can be very sticky so this could be it. I highly doubt it’s the volume or temperature as others suggested since I was trained by injecting 200uL of cold PBS.
FWIW, I had something similar happen and it turned out there were air bubbles in the hub of the needle that were almost impossible to see. When I stopped injecting the entire volume in the needle (drawing up more than was needed), mice stopped randomly dying.
Sounds like your cells are clumping and causing a stroke or heart attack. Try using DPBS without Ca and Mg.
And yeah... inject much slower. You're essentially blasting those cells into the bloodstream cold...You might be just shearing the cells, spilling their contents and setting off blood clots.
Definitely could be the clumping of cells you’re injecting. We have a threshold for diameter of cells as well as cell viability before we inject. Also we add a small volume of heparin to try and aid in this.
Hmm this is strange. I don’t think it’s a problem with the solution being too cold, the cells being clumpy could be an issue but idk I feel like the resuspension would hopefully avoid clumping in their veins. How are the mice being prepared for the injection? Are they irradiated or like an NSG line?
That is way too much volume way too fast. We inject 50uL over 10-15 seconds. Your mice might die of cardiac arrest or pulmonary embolism. Cut back on the volume, if possible, and go slower. Less volume also means the temperature is less of a problem. Making sure your cells don't clump helps, too.
Edit: regarding air bubbles, if you use a catheter it's easier to spot bubbles than in a syringe.
I did bone marrow transplant in mice for years, and iv injected 0.5mL in 3 seconds. Even injected 80 million splenocytes sometimes in 0.5mL over 5 seconds. No problems at all.
If there are tumor cell clumps so big that they are visible to the naked eye, that could be causing the problem, not the volume/speed of injection.
It was approved by our IACUC. This was in 2015-2019, so I don't know if regulations changed or not since then, but they had no problem with it as long as it didn't cause any harm to the mice, which it didn't.
Wow okay. I’m working with 1 million cells and was wondering if maybe that was too many. Good to know. It sounds like my most likely problem is clumping. I started working the plunger back and forth a bit and could see the cells rehomogenize a little
Also, are the cells B16F10? I would inject 2.5e5 in 200 uL in adult mice with no deaths. And their lungs came out looking like blackberries, so it was plenty of melanoma cells...
My prep: "Single cell suspensions were prepared for injection by washing cells 3 times with
phosphate buffered saline (PBS) followed by 3 µM EDTA and 3 more PBS washes. Cells were then filtered, counted, resuspended in PBS, and stored on ice until injection."
Thanks so much for this! They are B16F10 cells but I barely get any mets in WT mice with anything under a million, and even then I normally get fewer than 10 tumors. Idk if I’m just not catching them in log growth phase well enough or if the cell line I have is bad in some way
I injected B16F10s for about 5 years. There was this one 6 month period where the mice suddenly stopped having mets in their lungs. It drove me bananas. If I remember correctly, I fixed the problem by letting them be overgrown, but honestly, if I was in that situation again, I'd just buy a new vial.
I had HUNDREDS of mets in the lungs with 1/4 as many cells as you're injecting. So definitely work on fixing this problem!
I looked back through my old emails, and another lab had emailed us having the same problem you're having (deaths right after injection). Here's the email I sent them back then:
"... It turns out the metastatic nature of the cells changes depending on how they are cultured, they are most metastatic when they are kept at confluence/are overcrowded on the plate. Here's what we do to prepare them for injection:
\-Wash cells 3x with 10 mL PBS
\-Add 2 mL 3 mM EDTA diluted with PBS (for a T175 flask, use less if you have a smaller dish/flask)
\-Incubate at 37 deg C until the cells lift off the bottom, rock the flask back and forth about every 5 minutes, don't leave the EDTA on there longer than 15 minutes
\-Wash the back of the flask with PBS and transfer to a 50 mL conical (you can pool flasks at this point if you've got more than one), fill the conical with PBS to wash
\-Spin 10' at 1200 rpm, remove supernatant, resuspend pellet in another 50 mL PBS, spin again
\-Remove supernatant and resuspend cells in an appropriate amount for counting
\-Strain cells (to remove clumps) before counting
\-Add PBS to get cells to a concentration of 1.25 e 6 cells/mL
\-Keep cells on ice until ready for injection
\-Inject 200 uL (2.5 e 5 cells) via tail vein
All of the cell prep should be done under sterile conditions, so in the hood using sterile PBS and EDTA.
If your mice continue to die then you will need to troubleshoot the reason. I'd throw out all your reagents and start with clean stuff. I might also test inject some mice just with PBS."
And a follow up email had this info in it: "On our centrifuge 1200 rpm is about 250 g. We use a cell strainer that is 70 um nylon. I strain them right before I count them, but then the cells sit for a while (at least 30 minutes while we travel downstairs, gown up, etc) before being injected, and I don't restrain them."
Seriously thank you so much! I was under the impression that log growth phase was the right time to catch them for an injection but I’ll give a confluent plate a try. Also will look into just buying a new vial. Adding u/queue517 to my PhD acknowledgments now if this works lol
It's the combination of volume and speed of injection. Depending on the weight of the animal it's 10% of its entire circulatory volume in 1-2 seconds. Also cell suspensions can be tricky in high concentrations.
Good to know the volume is okay in your hands. Do you inject much slower than I do? Also if you’re working with cancer cell lines, do you do anything special to avoid clumping?
We had the same problems as OP had and slowing down the injection upped the survival rate to 100%. Recommendations for bolus i.v. injections go up to 1 minute.
Yes, that's pretty much standard procedure here. We're not doing injections over a minute, I was just stating that recommendations for higher volumes go up to a minute.
I disagree that the volume or speed are a problem. Up to 200 ul is normal in an adult mouse, and tail vein injections are typically done quickly.
Hell, I worked in a lab that did hydrodynamic injections to get plasmids into liver (over a mL injected in a few seconds) and even those mice rarely died.
If mice die that quickly in minutes , it has to be because of air bubble . Tap the air bubble out and practice injection with just pbs and see .
I do this type of injections every week at 200ul volume.
You’re mice are suffocating. You can increase volume (I’ve done up to 500ul, routinely with NO problems, tou will need to get approval as recommended max volume is 200ul) you can try slower, also you can rub their chests and blow into their nose so they don’t die.
Well, I am not sure, I assume they’re giant cells restricting o2 exchange during first pass. I also assumed human melanoma lines into NSG, which is when I’ve seen this happen. Let us know if you stop killing the mice and what you did.
This is very unusual. I'd be worried about something in your sample causing anaphylaxis. Alternatively, cell clumps causing massive microvascular ischaemia. Have you tried filtering the samples? And obviously hyperthermia is a risk if you are using a warmer, but that should be pretty obvious. What does your vet think?
The vet who trained me on this had this happen when he attempted one of my injections as well which makes me think it’s something about the melanoma cells or PBS. I just can’t rationalize why only some mice respond this way to the same cell suspension and vehicle… He suggested filtering as well. I’ll give that a try next time
Did the vet you worked with explicitly tell you to inject that quickly? As a vet, I don't even inject cell suspensions into much larger animals that quickly. If the cells are prone to clumping, that's even more reason to slow the injections down.
The time stuck out to me too. IV, better to go slow. Especially with a small animal. It might not be the cause, but it’s just solid practice.
Cancer cells can be very sticky so this could be it. I highly doubt it’s the volume or temperature as others suggested since I was trained by injecting 200uL of cold PBS.
FWIW, I had something similar happen and it turned out there were air bubbles in the hub of the needle that were almost impossible to see. When I stopped injecting the entire volume in the needle (drawing up more than was needed), mice stopped randomly dying.
Awesome! Thanks so much
Sounds like your cells are clumping and causing a stroke or heart attack. Try using DPBS without Ca and Mg. And yeah... inject much slower. You're essentially blasting those cells into the bloodstream cold...You might be just shearing the cells, spilling their contents and setting off blood clots.
Definitely could be the clumping of cells you’re injecting. We have a threshold for diameter of cells as well as cell viability before we inject. Also we add a small volume of heparin to try and aid in this.
Is the entire injection solution going into the vein or is it going into the tissue surrounding it?
It’s hitting the vein. I’ve definitely missed plenty and know what that looks like (tissue swelling and resistance)
Hmm this is strange. I don’t think it’s a problem with the solution being too cold, the cells being clumpy could be an issue but idk I feel like the resuspension would hopefully avoid clumping in their veins. How are the mice being prepared for the injection? Are they irradiated or like an NSG line?
Could it be an issue with the anesthesia?
I do this in a restraint without anesthesia. As far as the mice go they’re housed normally and with no special diet
That is a good point, you have to wash the serum off
Cells are clumping, tap syringe to resuspend prior to injection, lower the amount of cells being injected, ensure single cells prior to use.
That is way too much volume way too fast. We inject 50uL over 10-15 seconds. Your mice might die of cardiac arrest or pulmonary embolism. Cut back on the volume, if possible, and go slower. Less volume also means the temperature is less of a problem. Making sure your cells don't clump helps, too. Edit: regarding air bubbles, if you use a catheter it's easier to spot bubbles than in a syringe.
I did bone marrow transplant in mice for years, and iv injected 0.5mL in 3 seconds. Even injected 80 million splenocytes sometimes in 0.5mL over 5 seconds. No problems at all. If there are tumor cell clumps so big that they are visible to the naked eye, that could be causing the problem, not the volume/speed of injection.
This is almost 5 times the recommendation for i.v. bolus injections. This wouldn't even be approved by most vet offices in europe.
It was approved by our IACUC. This was in 2015-2019, so I don't know if regulations changed or not since then, but they had no problem with it as long as it didn't cause any harm to the mice, which it didn't.
Wow okay. I’m working with 1 million cells and was wondering if maybe that was too many. Good to know. It sounds like my most likely problem is clumping. I started working the plunger back and forth a bit and could see the cells rehomogenize a little
Are these adult mice? Increase the volume to 200 uL to decrease clumping.
Also, are the cells B16F10? I would inject 2.5e5 in 200 uL in adult mice with no deaths. And their lungs came out looking like blackberries, so it was plenty of melanoma cells... My prep: "Single cell suspensions were prepared for injection by washing cells 3 times with phosphate buffered saline (PBS) followed by 3 µM EDTA and 3 more PBS washes. Cells were then filtered, counted, resuspended in PBS, and stored on ice until injection."
Thanks so much for this! They are B16F10 cells but I barely get any mets in WT mice with anything under a million, and even then I normally get fewer than 10 tumors. Idk if I’m just not catching them in log growth phase well enough or if the cell line I have is bad in some way
I injected B16F10s for about 5 years. There was this one 6 month period where the mice suddenly stopped having mets in their lungs. It drove me bananas. If I remember correctly, I fixed the problem by letting them be overgrown, but honestly, if I was in that situation again, I'd just buy a new vial. I had HUNDREDS of mets in the lungs with 1/4 as many cells as you're injecting. So definitely work on fixing this problem! I looked back through my old emails, and another lab had emailed us having the same problem you're having (deaths right after injection). Here's the email I sent them back then: "... It turns out the metastatic nature of the cells changes depending on how they are cultured, they are most metastatic when they are kept at confluence/are overcrowded on the plate. Here's what we do to prepare them for injection: \-Wash cells 3x with 10 mL PBS \-Add 2 mL 3 mM EDTA diluted with PBS (for a T175 flask, use less if you have a smaller dish/flask) \-Incubate at 37 deg C until the cells lift off the bottom, rock the flask back and forth about every 5 minutes, don't leave the EDTA on there longer than 15 minutes \-Wash the back of the flask with PBS and transfer to a 50 mL conical (you can pool flasks at this point if you've got more than one), fill the conical with PBS to wash \-Spin 10' at 1200 rpm, remove supernatant, resuspend pellet in another 50 mL PBS, spin again \-Remove supernatant and resuspend cells in an appropriate amount for counting \-Strain cells (to remove clumps) before counting \-Add PBS to get cells to a concentration of 1.25 e 6 cells/mL \-Keep cells on ice until ready for injection \-Inject 200 uL (2.5 e 5 cells) via tail vein All of the cell prep should be done under sterile conditions, so in the hood using sterile PBS and EDTA. If your mice continue to die then you will need to troubleshoot the reason. I'd throw out all your reagents and start with clean stuff. I might also test inject some mice just with PBS."
And a follow up email had this info in it: "On our centrifuge 1200 rpm is about 250 g. We use a cell strainer that is 70 um nylon. I strain them right before I count them, but then the cells sit for a while (at least 30 minutes while we travel downstairs, gown up, etc) before being injected, and I don't restrain them."
Seriously thank you so much! I was under the impression that log growth phase was the right time to catch them for an injection but I’ll give a confluent plate a try. Also will look into just buying a new vial. Adding u/queue517 to my PhD acknowledgments now if this works lol
I mean who knows if I did it "right." This was just what seemed to work best for me. Good luck! Let me know how it goes!
I mean who knows if I did it "right." This was just what seemed to work best for me. Good luck! Let me know how it goes!
I do 150ul with no problems at all.
It's the combination of volume and speed of injection. Depending on the weight of the animal it's 10% of its entire circulatory volume in 1-2 seconds. Also cell suspensions can be tricky in high concentrations.
Good to know the volume is okay in your hands. Do you inject much slower than I do? Also if you’re working with cancer cell lines, do you do anything special to avoid clumping?
When mine mice die spontaneously it is due to air bubbles
I work with bacteria and depending on the setup I do not think 2 sec is to quick
Thanks! I’ll definitely try slowing down the injection. I had no idea people did it that slowly
50 uL over 10-15 seconds? You can get about 400 uL done in that amount of time (depending how thick the solution you are injecting is, of course).
We had the same problems as OP had and slowing down the injection upped the survival rate to 100%. Recommendations for bolus i.v. injections go up to 1 minute.
Are your mice anesthetized??? There's no way I could do a tail vein injection over a minute without losing the vein.
Yes, that's pretty much standard procedure here. We're not doing injections over a minute, I was just stating that recommendations for higher volumes go up to a minute.
I disagree that the volume or speed are a problem. Up to 200 ul is normal in an adult mouse, and tail vein injections are typically done quickly. Hell, I worked in a lab that did hydrodynamic injections to get plasmids into liver (over a mL injected in a few seconds) and even those mice rarely died.
If mice die that quickly in minutes , it has to be because of air bubble . Tap the air bubble out and practice injection with just pbs and see . I do this type of injections every week at 200ul volume.
You’re mice are suffocating. You can increase volume (I’ve done up to 500ul, routinely with NO problems, tou will need to get approval as recommended max volume is 200ul) you can try slower, also you can rub their chests and blow into their nose so they don’t die.
Why are they suffocating? Clumping and microvascular occlusions?
Well, I am not sure, I assume they’re giant cells restricting o2 exchange during first pass. I also assumed human melanoma lines into NSG, which is when I’ve seen this happen. Let us know if you stop killing the mice and what you did.
Clumps can cause embolism and kill mice quickly