I have used only Advanced Instruments osmometers and they are great/ reliable.

As for the solutions, I wouldn't make bath everyday, in fact we use bath for weeks on end. The internal (if you go whole cell) is what we make everyday.

We make a regenerative stock X10 with GTP ATP, etc..and aliquot it to freeze it. That way, you only take ~150 uL of it everyday. We make a base solution with HEPES, K salts, TEA etc.. and keep that in the fridge.

On day of exp, just mix the base solution with the regenerative stock.

Thanks Wei and Olivier for the promptly responses! 

@Wei: I agree that the osmolarity should not change if we make solution the same way. The reason why I am thinking of doing it is because I make 10x solution of the bath and make my 1x from then. I am just a bit afraid that the 10x could evaporate over time, hence the osmolarity needs to be monitored and adjusted accordingly. :) Thanks for the recommendation! 

@Olivier: Yup, the Advanced Instruments osmometers are really nice. :) Interesting to hear that you actually are doing the reverse of what most people I know are. Don't you concern about the precipitation of calcium or evaporation of your solution? This still makes sense to me since I just checked  a bath solution that was made 10 days ago, and the osmolarity still the same. :) Yet I am curious why you choose to make the internal fresh everyday?  Thanks again for the help! 

I use Osmomat 030 from Gonotec. They discontinued this model, but sell similar Osmomat 3000. Neurons are quite sensitive to osmolarity, so we check every bath solution we made. Also, we measure every pipette solution, but then we aliquot and freeze them and we do not re-chech osmolarity after defrosting.

I've used both Wescor 5520 and Wescor Vapro 5600 and both are good instruments, but thinking about the "small lab" budget maybe 5520 may be represent a more suitable choice for you.

Regarding solutions what I can recommend (and in my case it works both for acute brain slices and neuronal/induced neuronal cells cultures) is:

1) prepare internal solution, measure osmolarity and store it frozen as single usage aliquots;

2) once a week prepare your extracellular bath solution as 1X (so at it's final experimental concentration) adjusting osmolarity to match your cell culture medium, sterile filter it using 0.45um filter units and store it at +4°C up to 1 week in 100 ml aliquots.

Please keep in mind that you have to adjust your solutions in order to have a difference of 5 to 10 mOsm/kg between your extracellular and intracellular (intra>extra) in order to ease the Gigaseal formation.

Please feel free to contact me if you need any help.

Good luck.

In my experience,

Freezing the internal solution at -20oC is doable in small aliquots (~1mL). It can be stored for over 6mos.  When thawing, allow the solution to than get to near room temp. and then keep on a bed of ice if you have ATP/GTP in your solutions.  It should be good for the day of patching.

For bath solutions, I personally make 1-2x a week.  And store at 4oC when not using it.

Unfortunately, I am not familar with our osmometer brand.  I am sure  the above suggestions may help you out with that.

Hi all,

I am so overwhelmingly happy with all of the suggestion and advice! Looks like Wescor 5600 is the way to go for osmometer. I just requested the quote from them. :) 

For the internal solutions, I am pretty much doing quite the same thing that you guys are. Yet I have not been making fresh external very regularly and also not been aliquoting it to 100 ml aliquots. Really good to learn.  

Thanks again everyone! You are so helpful! 

we use also the wescor 5600. very convinient

We check the osmolarity when we  prepare new internal solution / usually measure osmolarity of external sol. also (a difference of  10 to  20 mosm is quite good,  better for access resistance). store @ -20°C for six months (aliquot of 1ml).

We prepare our extracellular bath solution as 10X  adjusting osmolarity and pH (need to verify and adjust pH when you dilute it to obtain the  final experimental concentration / also take care about the calcium conc. / possible precipitation) and store it at -20°C up to 4 week in 50 ml aliquots (falcon tube).

I've been using Advanced Instruments for the last 17yrs.  About 8 years ago I replaced the one we inherited from a previous lab with the model 3300....works fantastically!

As far as solutions, external solutions I make up 1x about 4 liters at a time, sterile filter and store at 4C for weeks without any issues.  Internal solutions, like many suggestions above, are stored -20C in small 1ml aliquots and are kept sometimes up to a year without any changes in properties.  I do however, store the internal in eppendorf tubes which are then stored in a sealed jar.

Good Luck!

Thanks Chris and Ann! 

I have been using Vapro Osmometer 5520 for over 10 years, but this product has been discontinued and has been replaced by the Vapro 5600, which costs more but is more convenient to use.

Similtar to Chang- Ning Lui I have using Vapro Osmometer 5520 for several years. I normally check osmolarity once a week . Internal solution is made in small amounts 100-200ml , aliquoted in 5 or 1 ml  and stored at -20. Normally i change solutions every two days. 

I have found that that stuffed osmolarity is the most common easily detected problem leading to unstable recordings in dissociated neurons and cell lines - so I measure it every day.  Makes sense when making externals from 10x, and also internals, even if they are from the same stock of frozen aliquots.  5 minutes can save a half day of frustration.  Of course, people were patching without ready access to osmometers for years, so it isn't essential...but if an osmometer is available, then why not make you life easy.

For what it is worth, I would make about 100 mls of basic internal, keep this at 4oC then add ATP/GTP to 10-15 mls and freeze in 1 ml aliquots.  After 2 weeks I would chuck'em and make fresh.  3 weeks is probably OK, but a regular turnover stops problems before they have a chance to start.  And osmoming solutions with salts like TEA is essential - they have really variable water content, both when purchased, and after sitting around on the shelf.

Thanks all! 

@Mark: so you will trash the internal after 3 weeks? Wah. It is quite a pain to make so has been keeping the stock w/o ATP/GFP for a few months, and add ATP/GTP into 10 ml then aliquot it into 200 ul and store at -20oC. I plan to use these aliquots for a month or two... 

Hi,

It depends, if you have enough money advanced instruments model 3320 is the one i will recommend,   the volume to check the osmolarity is 20ul. they also have models with different price range.

another  one is  from Knauer germay, semi micro osmometer model k 7400. Both osmometers  worked pretty well for me.

thanks

pradeep

Thanks Pradeep!

I used a Semi-Micro Osmometer from KNAUER (http://www.knauer.net/de/produkte/produkttypen/osmometer.html). Different pipette solutions were prepared with adjusted pH and osmolality in 100 ml Merck bottles and stored in the freezer. Each solution, depending of the experiment I wanted to do, was warmed every day early morning in a warm bath (28 Celsius). Bath solution were prepared in 1-liter bottles and stored at 4 grad Celsius in the refrigerator. Remember to control every day pH and osmolality, they are quite critical for patch-clamp recording or any other in vitro experiments.

Hello, I see many of you here are experienced users of Advanced Instruments osmometer, so forgive me to ask my question here. I have a question about using the Advanced Instruments model 3300. We have the osmometer in the lab but the 20uL sampler is missing. I have no experience using the machine. Do I have to use the special sampler (Fisher Scientific selling it for nearly $700), or it is possible to substitute with a Hamilton syringe or something similar? Thank you very much.

Just to share the reply from the company to my question: "Unfortunately you would not be to use another companies’ pipette with this instrument. The reason for this is that the sample is tested INSIDE the sample tip; nothing is actually “injected” into the instrument. The pipette is specially designed so that the measuring Probe can accurately monitor the sample temperature inside the Pipette tip. Following the supercooling stage, a solenoid strikes the sample tip to induce freezing, and if any other pipette is used then the sample would not freeze properly."