CELL AND MEDIUM COLLECTION

All precautions needed were taken to avoid any contamination of the candidate cells and collected medium. Firstly, all glassware was well washed and autoclaved and all plastic ware used was as a brand new and sterile. Secondly, the UV-C lamp in the laminar flow hood was turned on to sterilise the surfaces for at least 15 minutes. Thirdly, the cell culture hood was wiped by1% Vircon solution, and wiped by 70% ethanol as well, to make sure a clean environment for cell and medium collection was obtained. Fourthly, collection tubes were completely sprayed with 70% ethanol and opened inside a sterile cell culture hood. In aseptic way, cell lysate or extract and medium were transferred into RNase-free 2 ml centrifuge tubes or sterile 1.5 ml Eppendorf tubes (Eppendorf, UK).

2.6.1 Cell culture medium collection 2.6.1.1 The practical basis

Cell culture medium was collected prior to cell collection for measurement of secreted proteins as conditioned medium is a good source of secreted proteins by the cells. ELISA was used to measure secreted protein concentrations after a given treatment to study changes in the secretion pattern.

61 2.6.1.2 Medium collection method

Equipment

Pipette and sterile pipette tips RNase-free 2ml centrifuge tubes Sterile 1.5 ml Eppendorf tubes Sterile centrifuge tubes

The steps were carried out as follows: once the cells were ready to study, Either the preadipocytes or adipocytes conditioned medium was collected into 1.5 ml Eppendorf or RNase-free 2 ml centrifuge tubes by taking just 1 ml from each well. The collected medium was stored at -80 ºC until further use.

2.6.2 Cell collection for Total RNA extraction 2.6.2.1 The practical basis

A strong denaturing chemical compound used to denature protein is guanidinium thiocyanate, which is able to efficiently breakdown cells, denature protein and inactivate RNase. Guanidinium thiocyanate is a key reagent in the TRIzol® solution (Invitrogen, Paisley, UK). To extract total RNA from preadipocytes or adipocytes, guanidinium thiocyanate-phenol-chloroform extraction was used based on the method described by Chomczynski and his colleague (Chomczynski and Sacchi, 2006). Chloroform, as detergent, binds to protein and lipids of the cell membrane dissolving them as non-aqueous compounds. Thus, it disrupts cell membrane by breaking bonds that hold the membrane together. RNA and DNA are aqueous compounds. As for phenol, it is used for separating the RNA aqueous supernatant from other phases that contain DNA and non-aqueous compounds.

2.6.2.2 Reagent and equipment Cell culture in plates

TRIzol® reagent (Contains phenol and guanidine isothiocyanate) RNase-free 2ml centrifuge tubes

Pipette and sterile pipette tips Rocking platform

62

After the cell medium were collected or discarded, 500 µl of TRIzol® solution was added into each well of the plate which was then shaken by using a rocking platform for 5 minutes to detach cells. After cells have detached, the cells suspension was pipetted up and down to ensure that all cells were mechanically dislodged. Then from each well the mixture was collected into RNase tubes and stored at -80 ºC until further investigation. The collected cells were used for RNA extraction (See section 2.9).

2.6.3 Collection of cell lysate 2.6.3.1 The practical basis

The cell collection was carried out for measuring biological molecules (intracellular proteins) through breakdown of candidate cells to allow isolation and extraction of the proteins which could be used for investigation. Lysis buffer was used to disrupt the cell membrane together with mechanical disruption by using pipetting for enhanced breakage. Most of the mammalian cells are generally fragile and can be disrupted by lysis buffer that contains sodium dodecyl sulphate detergent (SDS). SDS is considered as the main component of cell lysis buffer, where its function, in addition to lysing action, is to denature proteins into their primary structure. Protein extracts need to be collected as quickly as possible and stored at -80 ºC until use to reduce degradation. Therefore, it is highly recommended to add protease and phosphatase inhibitor cocktails to lysis buffer. Protease inhibitor acts to inhibit endogenous proteases which are released during cell disruption and degrade other proteins (Deutscher, 1990). The phosphatase inhibitor acts to inhibit the action of exogenous and endogenous phosphatases (Deutscher, 1990). These phosphatases may remove phosphate group from proteins which are phosphorylated by protein kinases, most likely at tyrosine or serine residues, during cell signal transduction episodes that are involved in several cellular processes such as cell growth, proliferation and cell apoptosis (Alberts et al., 2015). Moreover, there are many methods for lysing cells to extract total protein carried out depending on the protein of interest.

63 2.6.3.2 Reagent and equipment

Cell lysis buffer

50 mM Tris-HCl, pH 6.7 5% glycerol

2% SDS

Protease inhibitor cocktail (1:100) Phosphatase inhibitor cocktail (1:100) Cold autoclaved PBS

Pipette and sterile pipette tips RNase-free centrifuge tubes

2.6.3.3 Method

After the cell medium was aspirated, the monolayer of cells was washed two times with 1 ml of cold autoclaved PBS per well. 300 µl or 150 µl of lysis buffer was added into each well of a 6 well plate or 12 well plate respectively. Tapping the plate on the bench was needed to remove the attached cells from the bottom of the wells. The cells/lysis mixture was pipetted up and down to ensure that the maximum number of cells were collected and then transferred into RNase-free 2 ml centrifuge tubes and stored at -80 ºC to prevent protein degradation until further investigation.