# Optical Properties of Buffers and Cell Culture Media for Optofluidic and Sensing Applications
## 2 Materials and Methods  
### Buffers and CCM  
The liquids investigated in this experiment are commonly used in a wide range of cell culture procedures. Buffers are aqueous solutions consisting of a mixture of weak acid and a salt of this acid with a strong base, or a mixture of a weak base and a salt of this base with a strong acid. The investigated buffers are widely used in molecular biology, such as in electrophoresis, Western blots, cell suspensions, and procedures requiring DNA or RNA suspension. For the purpose of this study, we selected two widely used buffers, namely, Dulbecco Phosphate Buffered Saline (PBS) and TRIS-Borate-EDTA Buffer (TBE). TBE contains TRIS (tris(hydroxymethyl)aminomethane), boric acid, and ethylenediaminetetraacetic acid (EDTA), where EDTA is responsible for chelating divalent cations such as Mg\({}^{2+}\) in the solutions. In many biological procedures, TBE is issued to stabilize nucleic acids against enzymatic degradation [19]. PBS, in turn, contains NaCl, a phosphate buffer, and KCl. It has a pH of 7, which is neutral and allows for cell survival and performing analyses with RNA and proteins. These buffers do not contain any coloring ingredients/components.  
The two selected CCM, namely, Express Five\({}^{\rm TM}\) SFM (EXPRESS 5) and Dulbecco's Modified Eagle's Medium (DMEM), are widely used in many types of human and mammalian cell cultures or procedures, where protein products need to be overexpressed for further analysis [1; 2; 3; 5; 6]. The CCM have to contain specific nutrients such as glucose, sodium pyruvate (source of energy), specific egzogenous amino acids, NaHCO\({}_{3}\), and buffering salts [2]. They usually also have colored ingredients and specific pH-dependent dyes for visual monitoring of any adverse pH change [2; 3]. The investigated CCM contained a phenol red dye--Phenolsulfonphthalein. This dye can gradually transition from yellow in a pH of 6.2 (\(\lambda_{\rm max}\) = 443 nm) up to red in a pH of 8.2 (\(\lambda_{\rm max}\) = 570 nm); in a more alkaline pH (pH > 8.2), phenol red becomes bright pink. While using the CCM, the color change can indicate the loss of medium stability or change in composition. This may take place when the cultured cells excrete to the solution their metabolites, causing a change of pH. In cell culture practice, the color change may give information regarding whether or not the medium still contains the nutrients or other required substances, such as vitamins and growth factors, or if there has been over compaction of the culture [1; 2]. The main properties of the liquids considered in this work are summarized in Table 1.  
The buffers and CCM used in the study after first use were stored in a fridge for the entire analysis period (up to 8 months). Containers with the buffers and CCM were tempered for at least 30 min at room temperature prior to measurements. Fast heating up was avoided in order to maintain the CCM properties, as CCM contains carbonate ions (from NaHCO\({}_{3}\)) and fast heating could slightly change the pH to more alkaline. The measurements were performed for buffers and CCM as typically used, that is, without added reagents such as, for example, serum (e.g., fetal bovine serum), L-glutamine(in the case of High Five), or antibiotics (e.g., gentamycin). The use of the aforementioned additives depends on the type of cultivating cells and the planned analysis and vary considerably based on the cell culture protocol used. Measurements were performed without external change of the pH. Most of the CCM used for mammalian cell cultures have a pH of -7.4--the value is optimized for different cell types. Some cell types grow better in slightly different pHs (e.g., skin fibroblasts at 7.4-7.7, or some cancer cell lines at 7.0-7.4). An acidic pH can mimic the environment in the tissue surrounding the cancer lesion, as cancer cells have a much higher metabolic rate and expel many more protons through membrane-based mechanisms in order to regulate intracellular pH.