32k2

1. Aim
2. Materials
3. Methods
- 3.1. Day 0
- 3.2. Days 1-4
4. Results
5. Conclusions

1 Aim

Investigate whether the growth of the melanoma cell line B16-F1-Luc2-BR2 is impaired in the presence of beta-hydroxybutyrate (BHB), over the 5 day period of the experiment.
We begin with 32000 cells per 12-well plate.

2 Materials

B16-F1-Luc2-BR2 cells.
These were generated as follows: These were generated as follows:
- B16 cells were obtained from American Type Culture Collection (ATCC).
- To facilitate quantitative measurement of tumor growth, they were modified as follows:
  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.
- They were then injected into the right ventricle of a mouse.
  These animals were sacraficed when bioluminescence was detected in the brain.
- Cells metastatic to the brain were recovered put into culture.
(R)-(-)-3-hydroxy butyric acid sodium salt (Sigma-Aldrich, St. Louis, MO).
\(2 \times\) 12-well plastic culture plates (Falcon \textregistered)
12-well plastic culture plate (Falcon \textregistered)
Media: DMEM (Gibco \textregistered) +10% FCS + 600 ug/mL G418 + 1x glutamine. pH 7.4.
FCS = Fetal calf serum.
Trypsin/ EDTA.
Hemocytometer (manual and automated (Countess\texttrademark~by Invitrogen\texttrademark)
15 mL plastic centrifuge tube (VWR\texttrademark)
Other supplies:
- Trypan blue stain 10%.
- Eppendorf tubes (plastic, sterile).
- PCR tubes (for mixing cells and Trypan blue).
- Disposable slides for hemocytometer.

3 Methods

3.1 Day 0

The total cell count from the T-25 flask was 912x10³.
For the cells being grown with 10 umol/L BHB, add 0.01639g of (R)-(-)-3-hydroxy butyric acid sodium salt to the 50mL tube before the media and cells.
Seed 2x 12-well plates with 16000 cells in 1mL of media.
For 16000 cells, this is 16/438 = 0.035mL.
Add 13x 0.035mL = 0.46mL to a 50mL plastic tube (for mixing media with BHB) and top up to 13mL.
Mix well by pipetting up and down x20.
Add 1mL to each of the 12 wells.
Shake the plates gently to evenly distribute the cells.
Return to the incubator at 17:30
Incubate at 37C in 5% CO₂ for 24h.

3.2 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 x20 using glass Pasteur pipette to ensure the cells are thoroughly mixed.
Return plates to incubator.
Remove 10uL of cell suspension and add 10uL 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.

Table 1: Quantities of BHB required each day
Day	No. wells	Volume of media (mL)	BHB (grams)
1	9	10	0.01260
2	6	7	0.00882
3	3	4	0.00504

4 Results

   Day      Tx  Count
1    0 Control  32000
2    0 Control  32000
3    0 Control  32000
4    1 Control  34000
5    1 Control   6000
6    1 Control  14000
7    2 Control  30000
8    2 Control  26000
9    2 Control  34000
10   3 Control 180000
11   3 Control 124000
12   3 Control 118000
13   4 Control 462000
14   4 Control 534000
15   4 Control 310000
16   0     BHB  32000
17   0     BHB  32000
18   0     BHB  32000
19   1     BHB  20000
20   1     BHB   8000
21   1     BHB  10000
22   2     BHB  12000
23   2     BHB  22000
24   2     BHB  14000
25   3     BHB  62000
26   3     BHB 104000
27   3     BHB  74000
28   4     BHB 276000
29   4     BHB 252000
30   4     BHB 156000

4.1 Standard error

stdErr <- function(x) sqrt(var(x)) / sqrt(length(x))
library(plyr)
(df2 <- ddply(df1, c("Day", "Tx"), summarise,
             mean = mean(Count),
             SE = stdErr(Count)))

4.2 Plot

4.3 Day 4 t-test

with(df1[df1$Day==4, ], t.test(Count ~ Tx))
with(df1[df1$Day==4, ], t.test(Count ~ Tx, alternative="less"))

	Welch Two Sample t-test

data:  Count by Tx
t = -2.7454, df = 3.126, p-value = 0.06783
alternative hypothesis: true difference in means is not equal to 0
95 percent confidence interval:
 -442271.08   27604.41
sample estimates:
    mean in group BHB mean in group Control 
             228000.0              435333.3

	Welch Two Sample t-test

data:  Count by Tx
t = -2.7454, df = 3.126, p-value = 0.03391
alternative hypothesis: true difference in means is less than 0
95 percent confidence interval:
      -Inf -32497.28
sample estimates:
    mean in group BHB mean in group Control 
             228000.0              435333.3

4.4 Day 3 t-test

(with(df1[df1$Day==3, ], t.test(Count ~ Tx, alternative="less")))

	Welch Two Sample t-test

data:  Count by Tx
t = -2.5968, df = 3.38, p-value = 0.03569
alternative hypothesis: true difference in means is less than 0
95 percent confidence interval:
      -Inf -8117.604
sample estimates:
    mean in group BHB mean in group Control 
              80000.0              140666.7

4.5 Day 2 t-test

with(df1[df1$Day==2, ], t.test(Count ~ Tx, alternative="less"))

	Welch Two Sample t-test

data:  Count by Tx
t = -3.6556, df = 3.723, p-value = 0.01226
alternative hypothesis: true difference in means is less than 0
95 percent confidence interval:
      -Inf -5658.004
sample estimates:
    mean in group BHB mean in group Control 
                16000                 30000

5 Conclusions

B16-F1-Luc2-BR2 cells show impaired growth in culture in the presence of BHB at 10 mmol/L.