Our lab is on the fourth floor of a building without AC. Our protocol involves removing the brain, and immediately slicing in ice cold sucrose. Then we heat the slices back to physiological temp for 30 minutes in aCSF, which is followed by placing the slices in room temp aCSF for 30 minutes. From there we begin moving slices to the rig to begin recording. The slices on our rig are held at physiological temperatures while performing WCR's.
When patch clamping in winter, we get many good recordings. However, when there is heat during the Summer/Spring the lab space sits at, or more than, 29 degrees Celsius (84 degrees Fahrenheit). So during Spring/Summer "room temp" is very different from Fall/Winter. During the warmer days I have noticed a dip in cell/slice quality. And on cooler days the slice quality returns.
I was wondering if anyone has noticed the same. Temperature obviously is a factor for neuronal health, of course. However, I know some labs who hold their slices at physiological temperature throughout the entire removal, slicing, holding, patching process, and report great cell quality. I don't know if the fact that our lab is quite warm during half the year is why I'm noticing a dip in brain slice quality.
Has anyone noticed that room temperature variance matters? What temperature do you hold your slices at, following removal and slicing on a vibratome or whatever instrument your lab uses?
Hi Ian,
Perhaps, if after 30 min of slices recovering at 36C the slices would be kept at about 15C temperature they may survive better. To get about 15C we used a bucket full of ice. The slices in a glass beacker with oxygenating aCSF were positioned on top of the ice. This way the glass bottom of the beacker would be at near zero Celsius, while the sidewall of the beacker and bubbling oxygen will be relatively warm, which will result in the aCSF temperature at about 15C. To reduce cooling you can put sponge between ice and the beacker. More expensive option would be to use water bath with cooling capability.
Hi Ian,
Temperature is a tricky variable and its setting will change from prep to prep (my experience). I cannot say I experienced the same as you. What I noticed in my slices is that if the cutting solution isn't VERY cold, their quality afterwards isn't optimal. There's this webinar by Jonathan Ting:
https://youtu.be/A0pIWSWjlvY
He's done a lot different slicing varying several of the parameters. Maybe something here will help out.
Also, I like the idea of ice to cool down your slices (thanks Anatoli Kabakov!) especially in those hot summer days. That ought to help!
Best of luck,
Andre
Hi Ian,
Interestingly enough my lab also faces a similar problem, at higher room temperatures during then summer, anything above 28 degrees C, and it is nearly impossible to patch, my PI also agreed and found similar issues, that with varying and increasing temperatures patching ability changes dramatically. Optimal range for us is around 21-22 degrees C room temp, and the patches are great! Not exactly sure why, but our lab has been seeing this for years it seems.
Best of Luck,
Haushe
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