Hi All,
I have come across a few studies where inclusion of BSA in buffers relating to use with ROS markers, specifically H2DCFDA and DHE. I refer to Arthur et. al. paper titled, "ITAM receptor-mediated generation of reactive oxygen species in human platelets occurs via Syk-dependent and Syk-independent pathways." as an example.
I am utelizing H2DCFDA, MitoSox Red and DHE for a number of ROS generation assays in my platelet work and am inquiring as to why BSA inclusion is included in some studies and not others? Is there a specific reason as to why inclusion of BSA is important?
Many thanks
Hi Evan
Most researchers include BSA in their staining/suspension buffer for FACS as BSA keep the cells happy and prevent clumping.
With best regards
Shubhankar
We use BSA when are studying organelles to prevent agregations, but not in crude extracts.
BSA contains nutritional factors thus prevent cells feeling hungry. Also BSA prevents cell to cell adhesion
Hi Evan,
It is well known that BSA or FBS actually protects cells from ROS effect. In some studies BSA is included as a part of the buffer composition to protect the cells from ROS generation due to other cells or other external sources. This will be useful in studies like yours where you can negate the ROS generation other than your interested source.
Where cells are involved. we use complete growth medium. Sometimes, to keep the pH constant in the absence of carbon dioxide, we will use a HEPES-buffered system to keep the pH where it should be. For studies involving ROS formation, the idea is to approximate growth conditions, so the cells are not dying.
Yes, you add BSA or FBS in buffer /solution for keep a condition as the same as in circulation that you have O2, CO2 for constant pH buffering system
Determination of endogenously generated reactive oxygen species (ROS)
Endogenous ROS production in platelets was determined according to the method of Lopez et al. [25] with slight modifications using CMH2DCFDA, a ROS-sensitive fluorescent probe. PRP as well as washed platelet suspensions were independently treated with calcium ionophore (A23187, 10 µM) as agonist. For inhibition studies, pre-loaded platelets with agonist were incubated with 4f in increasing doses (0–100 µM) and the final volume was made up to 200 µL with HEPES-buffered saline [HBS, 145 mM NaCl, 10 mM HEPES, 10 mM D-glucose, 5 mM KCl, 1 mM MgSO4 and supplemented with 0.1% bovine serum albumin (BSA), pH 7.45] and incubated at 37°C for 1 h. The control (untreated) and treated platelets were then incubated with 10 µM CMH2DCFDA for 30 min at 37°C, fluorescence was recorded using Varioskan multimode plate reader (Thermo Scientifics, USA) by exciting the samples at 488 nm and measuring the resulting fluorescence at 530 nm." please refer to this important research article : PLoS One. 2014 Sep 19;9(9):e107182. doi: 10.1371/journal.pone.0107182. eCollection 2014.
A new ibuprofen derivative inhibits platelet aggregation and ROS mediated platelet apoptosis.
Rakesh KS1, Jagadish S1, Vinayaka AC1, Hemshekhar M2, Paul M2, Thushara RM2, Sundaram MS2, Swaroop TR1, Mohan CD1, Basappa3, Sadashiva MP1, Kemparaju K2, Girish KS4, Rangappa KS1."
BSA scavenges ROS, when BSA is present in your experimental medium you will likely underestimate the amount of ROS being formed. At least for DCFH2DA, cells can be exposed for ~30min, so I would advise using PBS instead of culture medium.
See articles below for more advice on your redox assays involving DCFH2DA:
https://www.researchgate.net/publication/313901176_Preparation_and_Practical_Applications_of_2%277%27-Dichlorodihydrofluorescein_in_Redox_Assays
Good luck!
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