IANH Protocol for testing of metal oxide NP in mammalian tissue culture cells to study the Cell viability based on an oxidative stress paradigm
C. Biological assays on RAW 264.7 (murine macrophage cell line):
C.1. Cell culture and co-incubation with NP
All cell cultures are maintained in 25 cm2 cell culture flasks, in which the cells were passaged at
70-80% confluency every 2 days. RAW 264.7 cells (ATCC # TIB-71) are cultured in Dulbecco’s
Modified Eagle Medium (DMEM) (Invitrogen, Cat. # 11995-065) containing 10% (10:1
mixture) FCS (Fetal Calf Serum) (Gemini Cat.# 100-106), 100 U/ml penicillin (Invitrogen Cat.#
10378-016), 100 μg/ml streptomycin (Invitrogen Cat.# 10378-016), and 2 mM L-glutamine
(Invitrogen Cat.# 10378-016). BEAS-2B (ATCC # CRL-9609) cells are cultured in BEGM
(Lonza Cat. # CC-3171) in type I rat tail collagen-coated flasks or plates.
All the NP solutions are prepared fresh from stock solutions (10 mg/ml). The stock solutions are
made up from dry powder unless already in solution. Depending on the volume concentration of
particles in solution, an appropriate volume of WFI grade H2O is added to the appropriate
volume of particles. Dry powder NP are weighed using a chemical scale (Mettler Toledo Cat.#
AL54) that can weigh out to 100 μg of material and brought up at a concentration equal to 10
mg/ml using cell WFI grade H2O (Irvine Scientific, Cat.# 9309). For cell culture experiments
this stock is vortexed (or sonicated using Sonicating Water Bath) immediately before diluting
into complete cell culture medium (that contains 10% fetal calf serum). For instance, a 50 μg/ml
exposure dose is made by adding 5 μl of the NP into 1 ml of complete culture medium. The NP
suspension is then sonicated for 10 seconds prior to cellular exposure assays.
Proposal: For subculture, for RAW 264.7 cells, pippetting is enough because they are loosely attached; for BEAS-
2B cells, collect cell using trypsin/EDTA and pipetting. No scraping is needed.