The process of the preparation of phosphate buffer saline Essay

3.1 Material and reagent
3.1.1 Plant material
Gynura procumbens were purchased from HERBagus Sdn. Bhd. Penang, Malaysia. The Fresh leaves were harvested, dried in oven at 40degreesC and milled into powder.
3.1.2 Reagent
Ficoll (M.W 400,000) (Nacalai Tesque), Rosewell Park Memorial Institute (RPMI 1640) (Lonza), CLI-095 (InvivoGen), Lipopolysaccharide (LPS) from E.coli 0157 (Nacalai Tesque), Polymyxin B sulphate (Nacalai Tesque), Screening Standard Human Magnetic Assay 5 plex kit, Human Magnetic Premixed Microparticle Cocktail, Human Premixed Biotin-Ab Cocktail, Streptavidin-PE Concentrate, Diluent RD2-1, Calibrator Diluent RD6-52, Wash Buffered Concentrate
3.1.2.1 Preparation of phosphate buffer saline
100 ml is phosphate buffer saline (PBS) pH 7.2 is prepared by dissolving 1 PBS tablet (MP Biomedicals, LLC France) in 100 ml of distilled water.
3.1.2.2 Preparation of Ficoll solution density 1.077g/ml
To obtain Ficoll with density off 1.077g/ml, 21%w/v of Ficoll is prepared. 21g of Ficoll (M.W. 400,000) powder (Nacalai Tesque) is dissolved in 100 ml of phosphate buffer saline. The concentration of Ficoll needed to produce the density of Ficoll 1.077g/ml are determined based on the graph of density against concentration below
3.1.2.3 Preparation of Trypan blue
5 ml of 0.4% Trypan blue solution were mixed with 10 ml of 1X phosphate buffer solution (PBS) in a reagent reservoir. 10 ml syringe with 0.22 um sterile syringe filter were used to draw the mixture out of the reservoir and transferred into a 15 ml Falcon tube.
3.1.2.4 Preparation of RPMI 1640 media
RPMI 1640 media was supplemented with Fetal Bovine Serum (FBS), L-Glutamine and Penicillin-Streptomycin mixture. In 500 ml of RPMI 1640 media, 50 ml FBS, 5 ml of 200 mM L-Glutamine (2mM) and 10ml of Penicillin-Streptomycin solution are added. The sterilised media is then filtered.
3.1.2.5 Preparation of CLI-095
1 mg CLI-095 in powdered form (1 mg/ml) was added into 1 ml of DMSO and vortex until it completely solubilized to obtain 1 mg/ml of stock solution. 1 mul of solubilized CLI-095 (1 mg/ml) is then added to 999 mul of sterile culture media (RPMI 1640) to obtain a woking solution with concentration of 1 mug/ml. The stock solution was aliquot and stored at -20C.
3.1.2.6 Preparation of lipopolysaccharide (LPS)
5 mg of Lipopolysaccharide (LPS) from E.coli 0157 in powdered form was weighted and diluted in 1ml of distilled water to obtain 5 mg/ml of stock solution. Vortex is used to completely solubilize the LPS. 2 mul of LPS is then added to 998 mul of distilled water to obtain a working solution with concentration of 10 mug/ml. The stock solution was aliquot and stored at -20C.
3.1.2.7 Preparation of polymixin-B sulphate (PMB)
5 mg of Polymyxin B sulfate in powdered form was weighed and diluted into 1 ml of distilled water and vortex until it is completely solubilized to obtain 5 mg/ml of stock solution. 2 mul of Polymyxin B sulphate is then added into 998 mul of distilled water to obtain a working solution with concentration of 10 mug/ml. The stock solution was aliquot and stored at -20C.
3.1.3 Instrument and equipment