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9.6. BIOLOGICAL EXAMINATIONS 9.6.1. Cytolytic and cytotoxic activities of aqueous graphene 9.6.1.1. Materials and methods Hemolytic assay Defibrinated horse blood (Oxoid Basingstoke UK SR0050C) was centrifuged (100 ×g for 5 min) and washed in 0.9 w/v% NaCl until the supernatant was colourless, when the harvested red blood cells (RBCs) were counted using a haemocytometer and diluted to 5x106 cells/ml. Diluted RBCs (100 μl) were added to replicates (n=4) of each concentration of each graphene preparation (100 μl) and made up to 1 ml with sterile 0.9 w/v% NaCl. After incubation at 37 oC for 30 min and removal of the RBCs by similar centrifugation, absorbance at 550 nm of aliquots (200 μl) of the supernatants was estimated (Tecan Safire plate reader). Controls of RBCs alone and RBCs treated with surfactant (5 w/v% Triton X-100) were taken as zero and 100 % lysis (respectively). Cell Culture Breast cancer cells (MCF-7) were grown at 37 oC under 5 % CO2 in Roswell Park Memorial Institute (RPMI) medium (Sigma Aldrich, UK. R8758), supplemented with 10 % Foetal Calf Serum (FCS, Sigma Aldrich, UK. F7524) and 1 w/v% penicillin/streptomycin solution (Sigma Aldrich, UK. P4333). Mouse macrophages (J774A.1) were grown in Dulbecco′s Modified Eagle′s Medium (DMEM) media (Sigma Aldrich, UK. D6429) supplemented with 10 v/v% FCS and a 1 w/v% penicillin/streptomycin. When confluent, cells were removed: MCF-7 by Trypsin EDTA treatment and J774A.1 by gentle cell scraping. Cells were harvested and washed in phosphate buffered saline (PBS) by centrifugation at 100 ×g for 5 min at room temperature and counted by haemocytometer. AlamarBlue Assay Both MCF-7 and J774A.1 cells were allowed to adhere overnight to the surface of micro-well plates (Costar 96well Cell Culture Cluster, Cat No. 3596) at a density of approximately 5000 cells per well, with replicates (n=6) , before treatment with graphene preparations diluted into growth medium. Double strength growth medium was prepared Appendix 248PDF Image | PRODUCTION AND APPLICATIONS OF GRAPHENE AND ITS COMPOSITES
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