Krebs Henseleit Solution
For 1 Liter
118mM NaCl 6.90 grams
4.7mM KCL 0.35 grams
1.2mM KH2PO4 0.16 grams
1.2mM MgSO4 0.14 grams
25mM NaHCO3 2.10 grams
11mM Glucose 1.98 grams
2.5mM CaCl2 0.28 grams (I forget to write here now its OK)
0.003mM EDTA.Na2.2H2O 0.0011grams
Please can any one tell me step by step to prepare Krebs Henseleit Solution because sometimes our krebs solution make problem for our experiment
Best reagard
You need to add 2.5 mM of CaCl2, once your listed components are dissolved.
Ca2+ is very important if you are working with smooth muscle. We usenKrebs-Ringer bicarbonate solution (KRB) of the following composition (in mmol/L): 130 NaCl, 4.7 KCl, 1.17 Na2HPO4, 1.16 MgSO4, 24.0 NaHCO3, and 6 glucose, bubbled with a mixture of 5% CO2 and 95% O2, giving a pH of 7.4 after that you must add 2.5 mM of CaCl2 . We use Ca2+ quelators only when we want to buffer the Ca2+ concentration
My composition is really close to Reza's, but glucose included. The base (classical) Krebs (in mM):
NaCl: 118
KCl: 4.5
KH2PO4: 1.4
MgSO4: 1.2
NaHCO3: 25
glucose 11 mMol/L.
When everything dissolved add CaCl2: 1.4
Buble with carbogen & check the pH when its @ 37 C
The composition is different for high K or Ca free. I begin with classical Krebs for vessel adaptation in to the bath.
I add CaCl2 from the beginning, You try to get rid of Ca even with adding EDTA. For resting (adaptation period) why do you take the vessels in Ca free environment? If I needed this manuplation for smooth muscle, I switched the Krebs later on. I did not use Ca free Krebs for endothelium relaxation.
By the way many thanks for the discussion topik, we had an opportunity to see and think about the Krebs, from the very beginning of experimentation..
Thank you Adrian Gericke I add Cacl2 2.5 mM but I forget to write here
Thank you Reza Heidari for your knowledge
Thank you Veronica Milesi
Thank you Buket Demirci
You should add from 1 mM to 3 mM CaCl2, once you "finished" your buffer, but it depends on your aims. If you want working on cells contraction you have to add calcium into your buffer. Differently, you can use a CaCl2-free Krebs buffer solution.
The most common problem you could have is the calcium precipitation. So i suggest you to dissolve the CaCl2 seperately in half the volume of water, than add to the solution of the other chemicals under vigorous stirring. And remember that PH
Not oxygenate, gas with 95% O2, 5%CO2. The buffering requires the CO2.
Oh, and if you put the calcium in a little too quickly without stirring, it goes cloudy. Often bubbling with the Carbogen gets it back into solution. Gas not only the organ bath, but also the feeder reservoir, as well as any you have on the bench too keep it buffered and from going cloudy.
Hi,
When you gas the buffer with O2 and CO2, how long should it be done? Or should it just be done continuously throughout when you are using the buffer for your experiment?
we gas components (minus CaCl2 and MgSO4) for 15min on ice, then add CaCl2 & MgSO4 (ie Stock2) or the Ca will precipitate out due to temp&KH2PO4 conc.. see ref J Pharmacol Methods. 1985 Sep;14(2):147-55.
Calcium precipitation from mammalian physiological salines (Ringer solutions) and the preparation of high [Ca] media. MacConaill M.
I do Oxygenation by two bottle one O2% and Other Co2 %
we tried to mixed both of them deepening on PH on 7.4 by realtime determination of its PH
Hello Mudhir,
here is the standard Sigma K3753 Krebs-Henseleit Buffer with direct instruction. I have noticed 2 differences between Sigma standard and yours. First the Sigma recommends to add 0.373 g of calcium chloride dihydrate for each liter of solution being prepared but you showing 0.28 g and secondly, Sigma shows D-glucose 2 g in original package, and you are showing 1.98 g.
I think it is very important to also understand the nuances of preparation of this KH buffer as described by Sigma using step by step methodology at attached data sheet
Later, when you have standard solution ready you can use other above mentioned additions and publish with your modified solutions. Hope this helps,
Filip
Hello Mudhir,
after extensive search I have found full Krebs-Henseleit Buffer including all ingredients and pH 7.2 sold as is. I have made comparison slide for you. Please see it attached
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