I am trying to isolate neonatal rat cardiomyocytes and need to use CBFHH buffer. Does anyone have a recipe for a 10X concentration of this buffer. Thanks
Not knowing whether the given reagent amounts indicate 1x or 10x:
What about:
https://opus.bibliothek.uni-wuerzburg.de/files/4931/JanFiedler_PhD.pdf
p.38:
2.1.4.2 Cardiomyocyte preparation and fractionation of cardiac cells
Table 2.8: Cardiomyocte preparation medium CBFHH (Calcium- and Bicarbonate-Free Hanks´ solution with HEPES)
Component Stock Amount/l CBFHH [ml] Final concentration
NaCl 3.42 M 40 ml 137 mM
KCl 0.53 M 10 ml 5.36 mM
MgSO4*7H2O 81 mM 10 ml 0.81 mM
Dextrose 555 mM 10 ml 5.55 mM
KH2PO4 44 mM 10 ml 0.44 mM
Na2HPO4 34 mM 10 ml 0.34 mM
HEPES pH 7.4 0.206 M 100 ml 20.06 mM
Ad 1000 ml dH2O
Sterile filtration
Pen/Strep 1000x 1 1x
---------------------------------
2) cf: https://opus.bibliothek.uni-wuerzburg.de/files/2222/SabineMerkleDiss.pdf
p. 45:
CBFHH-Medium:
NaCl: 137 mM | KCl: 5,36 mM | MgSO4x7H2O: 0,81 mM |
Dextrose (Merck): 5,55 mM | KH2PO4: 0,44 mM | Na2HPO4x7H2O: 0,34 mM |
HEPES pH 7,3 (Chalibiochem) [NB:correct term=CALBIOCHEM] : 20,06 mM
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or: https://ediss.uni-goettingen.de/bitstream/handle/11858/00-1735-0000-0023-9947-D/Naim%20Kittana,%20PhD%20Thesis.pdf?sequence=1, you can find this Thesis publication also on RG @ https://www.researchgate.net/publication/269707044_Role_of_Secretory_Processes_in_Cardiac_Fibroblasts_in_Heart_Failure_Development_and_ProgressionRole_of_Secretory_Processes_in_Cardiac_Fibroblasts_in_Heart_Failure_Development_and_Progression :
p. 35:
Cell isolation
Calcium - and bicarbonate - free Hanks with HEPES (CBFHH) ( 1000 ml )
- 40 ml NaCl - stock (200 g/l)
- 10 ml MgSO4.H2O - stock (20 g/l)
- 10 ml KH2PO4 - stock (6 g/l)
- 10 ml Na2HPO4.2H2O - stock (5.97 g/l)
- 10 ml glucose dihydrate - stock(100 g/l)
- 100 ml HEPES - stock (47.66 g/l)
- up to 1000 ml distilled water, sterile by filtration.
- Adjust to pH 7.4 with NaOH
Good luck!
Thesis Role of Secretory Processes in Cardiac Fibroblasts in Heart ...
Thank you Wolfgang for your response and the links posted here. If it is not stated, I believe it should be 1x CBFHH buffer. Thanks for the recipe.
You're welcome, dear Bolanle, ....I think the your assumption the amounts and molarities given are 1x CBFHH is the most probable. Best wishes and the best!
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