Hi All,
I used media for humidity in my cell cutlure plate when incubating neutrophils, and one of the postdocs mentioned she's never heard of this. I've explained why, and humidity and 'have always done it' (not quite a good reason, I know) isn't good enough, and I just wondered if anyone a) does this as well, and b) why
Sophie
In a humidified incubator (these days almost all CO2 incubators are) it may be unnecessary.
Im not completely sure if I get your question right, but maybe you mean that you fill the outer wells of a plate with PBS/Media and only use the inside wells? In that case, this is in fact common and I also work with PBS filled outer wells especially in 96-Well plates.
Hi Patrick
Yes so I'd use 4 - 8 wells within a 24 well plate for my neutrophils, and put media in the outer wells, for humidity, especially for any overnight incubations. Other phd students in my lab do this, but I was told it wasn't necessary so a bit confused on this advice as its commonplace
From my experience it is not necessary to leave the outer wells in a 24 well plate unused. I frequently generated dendritic cells from monocytes in 24 well plates and had no problem with the outer wells. What is the volume per well that you use? You could simply measure the volume in the outer wells after an incubation period and see if there is any evaporation. From my experience less than 10 ul evaporate in 6 days of culture, but this may depend on your incubator.
Hi Patrick,
for neutrophil wells there would be about 500ul/well. in the outer surrounding wells I put 1ml in (which may be too much). our incubators are humidified, but as we're quite a busy lab, the incubators are frequently opened and closed, would this affect the humidity?
For 24 well plate the working volume per well is 0.4 -0.6 mL. When incubated at 37 Celsius and in presence of 95% (+/- 2%) relative humidity, loss of medium through evaporation is insignificant. We use these plates all the time and have not noticed any problem with our cultures and/or assays. Frequent opening will affect humidity; for every 30 second opening of the incubator, it takes at least 10 minutes to bring the humidity levels back to 95%. For an incubator that is opened and closed frequently, I would be more concerned about temperature and CO2 fluctuation that may significantly affect the cultures of sensitive cells.
Some excellent commentary is available here:
http://www.level.com.tw/html/ezcatfiles/vipweb20/img/img/20297/Corningcellculutretroubleshootinguide.pdf
I agree with Syed Ali. In a humidified CO2 incubator, with a large water reservoir in the bottom (we use 2 plastic mouse boxes 3/4 filled with distilled water and a copper pipe in the bottom (the Cu prevents growth of mold and bacteria, but do not put it in a metal container since it will react with Fe metals and eventually cause rust through)) and have no evaporation problem. When we use 96-well plates, because we take them out every day for measurements, we coat every plate, after filling all samples, with a sheet of BreatheEasy, which is permeable for air and CO2 but not water, and have no problems with pH, contamination, or evaporation.
Dear Christopher... could you please advise from which company or manufacturer do you get the sheets of Breatheasy, or if possible to advise what is it' cat. No.? Thanks a lot
Majid, I think that Thermo-Fisher Scientific carries it, but we usually get it from Diversified Biotech (800-796-9199; 65 Commerce Way, Dedham, MA, USA; www.divbio.com or [email protected]) Cat# BEM-1 for a box of 100 sterile sheets (list price $84/box). They come between 2 paper layers that can be removed (bottom side first, then spread over plate, use breyer (cat#BRAY-4000 or deluxe version BRAY-5000, both list at $19 each) to attach and seal wells, then remove top paper layer and put lid back on plate. We do all of this is a sterile vertical flow laminar flow hood. The Breathe-Easy is good between -80 oC and +100 oC. There are a few other versions, but they all have smaller temperature ranges.
Adding PBS to the outer wells is very common when doing assays in 96 well plates. The wells often have only 50 or 100ul volume and the greater evaporation that happens in the edge wells compared to the inner wells will change the concentration of the reagents in these outer wells compared to the inner wells making it more difficult to interpret the results. This also occurs in 384 well plates.
This effect on assays still happens using the modern humidified incubators.
When plates with 24 wells are used the evaporation effect is less likely to be noticeable since so many more wells are exposed to the edge (16 edge & 8 inner with 24 well plates vs 36 edge & 60 inner with 96 well plates) and greater volumes are used in the wells.
For growing cells it would depend on the sensitivity of the cells and how often the media is replaced.
We use 200 ul per well in 96 well plates and I think that the Breathe-Easy film and the humidity in the CO2 incubator solve the evaporation/concentration change problem.
We do fill the outer wells (96 wells plates) with 200 ul of either water or PBS. We perform incubations up to 10 days and without this supplemental humidity we may lose nearly half of the liquid volume. Even with the filled outer wells we lose some volume, but it is not much. Our incubator is also humidified.
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