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\title{16k}
\author{Chris Dardis}
\date{Friday 14th February, 2014}

\maketitle
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\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.\\
In previous work, we have shown that \num{8e3} cells/ 12-well plate
is insufficient to show sustained growth over 5 days.
We now use \num{16e3} cells.\\

\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.
\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{Day 0}

\formatdate{10}{2}{2014}

\begin{enumerate}
\item Cell counts from the T-25 flask are shown in table \ref{tab:ccd0}.
\subsubsection{Cell counts on Day 0}

<<Cell counts on Day 0>>=
@

\subsubsection{Mean}

<<Mean>>=
@

The mean from the \num{3} counts was thus \SI{340e3}{\mL}.

Seed a \num{12}-well plate with was \num{16e3} cells in \SI{1}{\mL} of media.\\
 For \num{16e3} cells, this is $\frac{16}{340} = \SI{0.047}{\mL}$.\\
 Add $13 \times \SI{0.047}{\mL} = \SI{0.61}{\mL}$ to a \SI{15}{\mL} graduated cylinder and
 top up to \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 and replace with \SI{1}{\mL} of new media.
\item For plates to be counted:\\
  Add \SI{1}{\mL} of trypsin to each in the row to be counted.\\
  Rock the plates.\\
  Remove the trypsin after \textless \SI{30}{\s}.
\item Add \SI{250}{\uL} of trypsin.
\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 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:data}.
\item Return plates to incubator.
\end{enumerate}

\section{Results}

<<Results>>=
@

\subsection{Standard Error}

<<Standard Error>>=
@

\subsection{Plot}

<<Plot>>=
@

\section{Conclusion}
A starting number of \num{16e3} cells appears more promising than \num{8e3} cells in terms of demonstrating sustained growth over the period of observation. See \texttt{8k.pdf} for comparison.

\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}

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