#+TITLE: 32k2
#+AUTHOR: Chris Dardis
#+DATE: \formatdate{24}{3}{2014}

* Aim

Investigate whether growth of a mouse melanoma cell line is impaired in the
presence of Beta-Hydroxy Butyrate (BHB).

* Materials

- *B16-F1-Luc2-BR2* cells.\\
  These were generated as follows:
  - B16 cells are available from ATCC.
  - These were were transfected in our lab with luciferase (to allow luminescence to be used a measure of cell numbers) along with a promotor.
  - They were then injected into the right ventricle of a mouse.\\
    These animals were sacraficed when they became symptommatic due to metastatic disease.
  - Cells metastatic to the brain were recovered and grown again in the same medium.
  These cells are the subject of this study.
- $2 \times$ 12-well plastic culture plates (Falcon)
- 12-well plastic culture plate (Falcon)
- Media: *DMEM* (Gibco) +10% FCS +
  $600\mathrm{microg/ml}$ G418 + 1x glutamine. pH 7.4.\\
  FCS = Fetal calf serum.
- Trypsin
- Hemocytometer (manual and automated (Countess by Invitrogen))
- 15 ml plastic centrifuge tube (VWR)
  * Trypan blue stain 10%
  * Eppendorf tubes (plastic, sterile)
  * PCR tubes (for mixing cells and Trypan blue)
  * Disposable slides for Countess

* Methods

** Day 0 

- The cell count from the T-25 flask was $912\times10^3$.
- For the cells being grown with $10\mathrm{micromol/L}$ BHB,
  add $0.01639\mathrm{g}$ of (R)-(-)-3-hydroxy butyric acid sodium salt to the $50\mathrm{ml}$ tube before the media and cells.
- Seed $2 \times 12$-well plates with $16\times10^3$ cells in $1\mathrm{ml}$ of media.\\
  For $16\times10^3$ cells, this is $\frac{16}{438} = 0.035\mathrm{ml}$. \\
  Add $13 \times 0.035\mathrm{ml} = 0.46\mathrm{ml}$ to a $50\mathrm{ml}$ plastic tube (for mixing media with BHB) and
  top up to $13\mathrm{ml}$. \\
  Mix well by pipetting up and down $\times 20$. \\
  Add $1\mathrm{ml}$ to each of the $12$ wells.
- Shake the plates gently to evenly distribute the cells.\\
  Return to the incubator at 17:30
- Incubate at $37^{\circ}\mathrm{C}$ in 5% CO_2 for $24\mathrm{h}$.

** Days 1-4

- For plates which are not being counted:\\
  Aspirate the old media and replace with 1ml of new media.
- For plates being grown in BHB, prepare the media was prepared by adding BHB as on day 0.\\
  The quantities of BHB required are shown in the table below.
- For plates to be counted:\\
  Add 2ml of trypsin to each in the row to be counted.\\
  Rock the plates.\\
  Remove the trypsin after < 30s.
- Add 250uL of trypsin.
- Incubate at 37 celsius 3min.
- Shake the plate to ensure all cells are dislodged from the base.\\
  Check with the microscope.
- 'Quench' the trypsin quenched with 250uL of media.\\
  Pipette up and down $\times 20$ using glass Pasteur pipette to ensure the cells are thoroughly mixed.
- Return plates to incubator.
- Remove 11uL of cell suspension and add 11uL Trypan Blue in a sterile PCR tube.\\
  Mix using a pipette.
- Place 10uL of the mixture into a sample well of the hemocytometer.
- Count the cells using the hemocytometer.\\
  These counts are shown in the table below.

#+CAPTION: Quantities of BHB required each day
#+LABEL: tab:bhb
| Day | No. wells | Volume of media (ml) | BHB (grams) |
|-----+-----------+----------------------+-------------|
| <c> | <c>       | <c>                  | <c>         |
| 1   | 9         | 10                   | 0.01260     |
| 2   | 6         | 7                    | 0.00882     |
| 3   | 3         | 4                    | 0.00504     |

* Results

#+NAME: Results
#+begin_src R :session *session* :exports results :results output :results verbatim :results pp
(df1 <- data.frame(Day = rep(rep(0:4, each=3), 2), 
                  Tx = rep(c("Control", "BHB"), each=15),
                  Count = c(rep(32e3, 3),
                  34e3, 6e3, 14e3,
                  30e3, 26e3, 34e3,
                  180e3, 124e3, 118e3,
                  462e3, 534e3, 310e3,
                  rep(32e3, 3),
                  20e3, 8e3, 10e3,
                  12e3, 22e3, 14e3,
                  62e3, 104e3, 74e3,
                  276e3, 252e3, 156e3)))
#+end_src

** Standard error

#+NAME: Standard error
#+begin_src R :session *session* :exports code :results output :results verbatim :results pp
stdErr <- function(x) sqrt(var(x)) / sqrt(length(x))
library(plyr)
(df2 <- ddply(df1, c("Day", "Tx"), summarise,
             mean = mean(Count),
             SE = stdErr(Count)))
#+end_src

** Plot

#+NAME: Plot
#+begin_src R :session *session* :exports results :results output :results graphics :file "f1.png"
library(ggplot2)
## plot with position dodge
pd <- position_dodge(.1)
ggplot(df2, aes(x=Day, y=mean, color=Tx)) +
    geom_errorbar(
        aes(ymin=mean-SE, ymax=mean+SE), width=.1, position=pd) +
    geom_line(position=pd) +
    geom_point(position=pd, size=3) +
    annotate("text", x=c(2, 3, 4), y=5e5, label="*", size=5) +
    ggtitle(expression(atop("Effect of BHB (10 mmol/lt) on growth of B16 cells", "* p<0.05 by t-test (1-sided)"))) +
    theme(plot.title = element_text(size=15)) +
    ylab("No. cells / ml")
#+end_src

** Day 4 t-test

#+NAME: Day4
#+begin_src R :session *session* :exports both :results output :results verbatim :results pp
with(df1[df1$Day==4, ], t.test(Count ~ Tx))
with(df1[df1$Day==4, ], t.test(Count ~ Tx, alternative="less"))
#+end_src

** Day 3 t-test

#+NAME: Day3
#+begin_src R :session *session* :exports both :results output :results verbatim :results pp
(with(df1[df1$Day==3, ], t.test(Count ~ Tx, alternative="less")))
#+end_src

** Day 2 t-test

#+NAME: Day2
#+begin_src R :session *session* :exports both :results output :results verbatim :results pp
with(df1[df1$Day==2, ], t.test(Count ~ Tx, alternative="less"))
#+end_src

* Conclusions

*B16-F1-Luc2-BR2* cells show impaired growth in culture in the presence of BHB.