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Lab Note #4 Second experiment

 Lab Note #4 Second experiment 

Since the previous result was not clear and cells did not get reduced as much as I thought, I decided to the second experiment by putting an increased concentration of PLA2 and 5FU on colorectal cells. 

Method:

1. I took plates with cells(WIDR and HCT15) out of a culture medium.

2. I got rid of liquid media using a suction machine. 

3. I put 500ul of TE buffer so that cells on the bottom of the plates float. After I put them on the centrifuge I waited for 5 minutes. (If the cells are on TE buffer too long, cells can die.) 

4. After 5 minutes, I slightly shook the plate with TE buffer and cells so that the cells are detached from the bottom of the plates. 

5. I put them on two 1.5ml tubes.

6. I put the tubes on a centrifuge that spin in a speed of 7500 rpm for 2 minutes. Then I can saw cells on the bottom of the tube separated from TE buffer. 

7. I sucked TE buffer carefully so that the cells don't get sucked up together. 

8. I put 200ul of RPMI and did a pipetting so that cells are mixed with RPMI evenly

9. I put RPMI + cells on 15ml tube (I made the liquid flow on the wall)

10. With a big pipette, I put 7 ml of RPMI and shook it up and down slightly.

11. I put 500ul of solution on a 24 wells plate. 

12. On the same 24 wells plate, I put PLA2 and 5FU according to the following concentration. 


 

 

1

2

3

4

5

6

 

PLA2

1500u/ml

0

5

10

20

40

50

u/ml

 

 

0

1.65

3.3

6.6

13.2

16.5

Ul

5FU

10mg/ml

0

200

400

600

800

 

uM

 

 

0

1.3

2.6

3.9

5.2

6.5

ul


After 72 hours, I checked the result using the same cell counting machine. 

 

HCT15

 

 

 

PLA2

0

5

10

20

Live Cell

2.20E+06

8.70E+05

1.07E+06

1.37E+06

Viability

93.3

93.2

94.8

96.9



 

HCT15

 

 

 

5FU

0

200

400

600

Live Cell

2.12E+06

2.02E+06

1.65E+06

1.49E+06

Viability

95.5

96.3

95.7

92

 

 

WIDR

 

 

 

PLA2

0

5

10

20

Live Cell

2.61E+06

3.55E+06

2.67E+06

2.44E+06

Viability

98.8

97.4

95.8

96.1

 

 

WIDR

 

 

 

5FU

0

200

400

600

Live Cell

2.15E+06

2.75E+05

5.70E+05

5.70E+05

Viability

97.6

80.6

86

86

Interpretation:

Half Life:

-22.3709

1709.09

10.60117

-824.385

I got minus half-lives, which proves that I could not get the result I wanted. 

So I decided to do the another experiment. 

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