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\title{8k}
\author{Chris Dardis}
\date{Monday 10th February, 2014}
\maketitle
\pagestyle{headings}
\tableofcontents
\section{Aim}
Establish the optimal number of B16-F1-Luc2-BR2 cells with which to
to seed a growth curve.\\
The goal is to demonstrate steady growth of the cells
during the 5 day period of the experiment.\\
This will be used to inform
future studies of agents which may inhibit growth.\\
We begin with \num{8e3} cells/ 12-well plate.
\section{Materials}
\begin{itemize}
\item \textbf{B16-F1-Luc2-BR2} cells.\\
These were generated as follows:
\begin{itemize}
\item B16 cells were obtained from American Type Culture Collection (ATCC).
\item To facilitate quantitative measurement of tumor growth, they were modified as described previously \cite{Abdel2012}. \\
The cells were stably transfected with the gene encoding luc2 (luciferase) using the pGL4.51 [luc2/CMV/Neo] vector (Promega Corp, Madison, WI) and FuGENEH 6 Transfection Reagent (Roche Applied Science, Indianapolis, IN) following conditions specified by the manufacturer.
\item They were then injected into the right ventricle of a mouse.\\
These animals were sacraficed when bioluminescence was detected in the brain.
\item Cells metastatic to the brain were recovered put into culture.
\end{itemize}
\item 12-well plastic culture plate (Falcon\textregistered).
\item Media: DMEM (Gibco \textregistered) +10\% FCS +
\SI{600}{\ug\per\mL} G418 + 1x glutamine. pH \num{7.4}.\\
FCS = Fetal Calf Serum.
\item Trypsin/ EDTA.\\
EDTA = EthyleneDiamineTetraAcetic acid.
\item Hemocytometer (manual and automated
(Countess\texttrademark~by Invitrogen\texttrademark).
\item \SI{15}{\mL} plastic centrifuge tube (VWR\texttrademark).
\item Other supplies:
\begin{itemize}
\item Trypan blue stain 10\%.
\item Eppendorf tubes (plastic, sterile).
\item PCR tubes (for mixing cells and Trypan blue).
\item Disposable slides for hemocytometer.
\end{itemize}
\end{itemize}
\section{Methods}
\subsection{Preparatory work}
On day -3, pass the cells at a ratio of $1:4$.\\
I.e. add \SI{0.4}{\mL} from the original flask to \SI{1.6}{\mL} of new media.\\
The T-25 flask is shown (prior to cell passage) in figure
\ref{fig:conf}.
\begin{figure}[h]
\subfloat[5x\label{fig:test1}]
{\includegraphics[width=.3\linewidth]{5xC}}\hfill
\subfloat[10x\label{fig:test2}]
{\includegraphics[width=.3\linewidth]{10xC}}\hfill
\subfloat[20x\label{fig:test3}]
{\includegraphics[width=.3\linewidth]{20xC}}
\caption{Visual confluency of cells in T-25 flask at various
magnifications}
\label{fig:conf}
\end{figure}
Count the cells. The results are shown in table
\ref{tab:count}.\\
Based on this the automated counter was thought to be reliable for this purpose.\\
For a seeding density of \num{1.2e4} per
well, approximately $12 \times \num{1.2e4} = \num{1.44e5}$ cells are required.\\
This is $\frac{1}{8}$ to $\frac{1}{10}$ of one T-25 flask.
\begin{table}[h]
\centering
\begin{tabular}{lccc}
Method & Trial & Cells/ mL & Cells in flask (\SI{2}{\mL})\\\midrule
Manual & 1 & \num{4.04e5} & \num{8.08e5}\\
& 2 & \num{4.72e5} & \num{9.44e5}\\
Automated & 1 & \num{4.3e5} & \num{8.6e5} \\
& 2 & \num{3.2e5} & \num{6.4e5}\\
\end{tabular}
\caption{Counts of viable cells by method}
\label{tab:count}
\end{table}
\subsection{Day 0}
\formatdate{3}{2}{2014}
\begin{enumerate}
\item Cell counts from the T-25 flask are shown in table \ref{tab:c2}.\\
The mean from the \num{3} counts is thus \SI{4.93e5}{\mL}.
\begin{table}[h]
\centering
\begin{tabular}{lc}
Trial & Cells per mL \\\midrule
1 & \num{3.92e5} \\
2 & \num{3.06e5} \\
3 & \num{7.82e5} \\
\end{tabular}
\caption{Cells counts on day 0}
\label{tab:c2}
\end{table}
Seed a 12-well plate with \num{8e3} cells in \SI{1}{\mL} of
media.\\
For \num{8e3} cells, this is
$\frac{8}{493} = \SI{0.0163}{\mL} = \SI{16.3}{\ul}$. \\
Add $13 \times \SI{16.3}{\ul} = \SI{0.212}{\mL}$ to a \SI{15}{\mL} centrifuge tube and add media to make a total volume of \SI{13}{\mL}. \\
Mix well. Add \SI{1}{\mL} to each of the \num{12} wells.\\
\item Shake the plates gently to evenly distribute the cells.\\
Return to the incubator at \formattime{17}{30}{00}.
\item Incubate at \SI{37}{\celsius}, in 5\% \ce{CO2} for
\SI{24}{\hour}.
\item Pass the remaining cells at $1:3$.\\
I.e. take \SI{0.5}{\mL} of media with cells from the
T-25 flask and add \SI{1.5}{\mL} of new media.
\end{enumerate}
\subsection{Days 1-4}
\begin{enumerate}
\item For plates which are not being counted:\\
Aspirate the old media aspirated replace with \SI{1}{\mL} of new media.
\item For plates to be counted:\\
Add \SI{1}{\mL} of trypsin/ EDTA to each in the row to be counted.\\
Rock the plates.\\
Remove the trypsin/ EDTA after \textless \SI{30}{\s}.
\item Add \SI{250}{\uL} of trypsin/ EDTA.
\item Incubate at \SI{37}{\celsius} for \SI{3}{\minute}.
\item Shake the plate to ensure all cells are dislodged from the base.\\
Check with the microscope.
\item 'Quench' the trypsin/ EDTA quenched with \SI{250}{\uL} of media.\\
Pipette up and down $\times 5$ to ensure the cells are thoroughly mixed.
\item Remove \SI{10}{\uL} of cell suspension and add \SI{10}{\uL}
Trypan Blue in a sterile PCR tube.\\
Mix using a pipette.
\item Place \SI{10}{\uL} of the mixture into a sample well of the hemocytometer.
\item Count the cells using the hemocytometer.\\
These counts are shown in table \ref{tab:c1to4}.
\item Return 12-wlll plates to the incubator.
\end{enumerate}
\section{Results}
\begin{table}[h]
\centering
\begin{tabular}{lcccc}
Day & Well & Trial & Cells/ mL & Mean of 2 readings \\\midrule
1 & A & 1 & \num{3.28e5} & \multirow{2}{*}{\num{193e3} } \\
& & 2 & \num{0.66e5} & \\
& B & 1 & \num{1.72e5} & \multirow{2}{*}{\num{148e3} }\\
& & 2 & \num{1.24e5} & \\
& C & 1 & \num{1.20e5} & \multirow{2}{*}{\num{159e3} }\\
& & 2 & \num{1.98e5} & \\
2 & A & 1 & \num{0.7e5} & \multirow{2}{*}{\num{78e3} } \\
& & 2 & \num{0.86e5} & \\
& B & 1 & \num{0.52e5} & \multirow{2}{*}{\num{63e3} }\\
& & 2 & \num{0.78e5} & \\
& C & 1 & \num{0.46e5} & \multirow{2}{*}{\num{79e3} }\\
& & 2 & \num{1.12e5} & \\
3 & A & 1 & \num{0.26e5} & \multirow{2}{*}{\num{33e3} } \\
& & 2 & \num{0.4e5} & \\
& B & 1 & \num{0.46e5} & \multirow{2}{*}{\num{56e3} }\\
& & 2 & \num{0.66e5} & \\
& C & 1 & \num{0.066e5} & \multirow{2}{*}{\num{33e3} }\\
& & 2 & \num{0.6e5} & \\
4 & A & 1 & \num{0.22e5} & \multirow{2}{*}{\num{13e3} } \\
& & 2 & \num{0.04e5} & \\
& B & 1 & \num{0.22e5} & \multirow{2}{*}{\num{15e3} }\\
& & 2 & \num{0.08e5} & \\
& C & 1 & \num{0.04e5} & \multirow{2}{*}{\num{10e3} }\\
& & 2 & \num{0.16e5} & \\
\end{tabular}
\caption{Cells counts day 1 - 4}
\label{tab:c1to4}
\end{table}
\section{Code}
\subsection{data}
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\subsection{stdError}
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\subsection{plot}
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\section{Conclusion}
Although the cells show vigorous growth initially, this is not sustained over the 5 day period of observation.
\bibliographystyle{plain}
\begin{thebibliography}{99}
\bibitem{Abdel2012}
Abdelwahab, Mohammed G., et al. The ketogenic diet is an effective adjuvant to radiation therapy for the treatment of malignant glioma. PloS one 7.5 (2012): e36197.
\end{thebibliography}
\end{document}