i just want to know if anyone knows the safe dose of minocycline that can be used in neuroblastoma cell line such as N2A? i did use in vivo but dont know much about invitro use...will be thankful for any references please.
Dear Syed,
I have copied some paragraphs from the following paper which I think cover the answers to your question:
Journal of Neurochemistry
Volume 105, Issue 5, Article first published online: 18 JAN 2008
Infection of mouse neuroblastoma cell line with JE virus, LDH assay, and immunoblot
Mouse neuroblastoma Neuro2a cells (N2a) were plated in 12-well plate at a density of 3 · 105 cells/well in 1 mL of medium, and were cultured for 18 h. Cells were pre-treated for 1 h with minocycline at concentration of 20 lM in serum-free media and then incubated with JE virus at different multiplicity of infection (MOI) for 1 h. The
virus was removed and the plates were then washed with 1x PBS to remove the unbound virus. The plates were further incubated in minocycline (20 lM) containing serum-free media at 37C for next 24 h. Then the supernatant was used for lactate dehydrogenase (LDH) assay to measure cell death (Promega) as described earlier
(Krady et al. 2005). For immunoblots, in another set of experiments N2a cells were plated. Following infection with JEV (MOI = 5) and subsequent minocycline treatment cells were processed for immunoblot analysis for Bax and Bcl-2 protein
(1 : 1000 dilution, Santa Cruz Biotechnology). Immunoblots were
performed as mentioned earlier. For TUNEL assay N2a cells were plated at a density of 1 · 104 cells per well of eight well chamber slides in medium containing
1% fetal bovine serum and exactly same four groups were examined as earlier (see LDH section). Assay was performed as mentioned earlier in the Material and methods section.
Intracellular staining by flow cytometry for JEV antigen Mouse neuroblastoma N2a cells were plated in six-well plate at a density of 5 · 105 cells/well in 3 mL of medium, and were cultured for 18 h. Cells were harvested in 1x PBS after the similar experiments as earlier mentioned (LDH assay). After two washes
with 1x PBS, cells were first fixed with BD cytofix solution (BD Biosciences) for 15 min. Then permeabilized by resuspending in permeabilization buffer (BD Cytoperm plus; BD Biosciences) and incubated at 22C for at least 10 min. Cells were washed twice in wash buffer (PBS containing 1% bovine serum albumin) then
resuspended in wash buffer at 1 · 106 cells per 100 lL. Primary antibody (JEV Nakayama strain; Chemicon) were added in 1 : 100 dilutions and incubated for 30 min at 22C. The cells were washed five times with wash buffer and pelleted. Then incubated with FITCconjugated secondary antibody for 30 min. After three times wash with wash buffer, finally, samples were resuspended in 400 lL FACS buffer and analyzed. Samples were analyzed on a FACS Calibur. Fold change was calculated by dividing the median fluorescence intensity of the infected and minocycline treated sample by that of the control sample (Krutzik and Nolan 2003). The percentage of population was calculated after gating the populations
on Dot plot in Cell Quest Software (BD Biosciences). Measurement of mitochondrial membrane potential Neuro2a cells were plated at subconfluent density. Twenty-four
hours later, cells were infected with JEV (MOI 1 : 5) and treated with minocycline as mentioned earlier. Later, the cells were incubated with 5 lM JC-1 fluorescence dye (Molecular Probes, Eugene, OR, USA) for 30 min and washed with PBS pre-warmed at 37C. Mitochondrial membrane potential was evaluated qualitatively under a Zeiss Axioplan 2 Fluorescence microscope (20· magnifications) using 568 nm filter (Swarup et al. 2007b).
To view the whole paper, please use the following link:
http://onlinelibrary.wiley.com/doi/10.1111/j.1471-4159.2008.05238.x/pdf
Hoping this will be helpful,
Rafik
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