Lab Note #2 The first experiment

 

Lab note #2

Experiment date: 2020.08.28

Material: phospholipase A2 from honey bee venom (Apis mellifera), 5-Fluorouracil, colorectal cancer cell. (WIDR and HCT 15). Pipette, plate, sucker (?),

 

Before I start an experiment, I observed cells using a microscope. I could see that compared to WIDR, HCT 15 has grown a lot and more compacted. The shapes of cells were both round. Several WIDR cells were compacted together, and this made the sizes of each cell different. (I could see that compacted cells are separated from each other after I put TE buffer.)

 

1. Making stock solution

*The concentration of stock solution is determined after reading several similar studies about PLA2 and 5 FU

Concentration 5 FU-1mg/ml / PLA2-1500u/ml

(I assumed the unit of PLA2 that I bought is __, the medium of the range mentioned in the selling site)

Method:

5-FU stock solution  

1.     Measure 0.015g of 5-FU powder

2.     Using a pipette, put Dimethyl sulfoxide (DMSO) in tube filled with 0,015g of 5-FU powder (organic solvent)

3.     Mix thoroughly with a machine about 5 minutes

PLA2 stock solution

1.     Put PBS in 1mg of PLA2

2.     Mix thoroughly with a machine about 5 minutes

Finally, I stored them in refrigerator.

 

2.

The purpose of experiment was to observe (1) the impact of PLA2 on colorectal cancer cells that have EGFR mutation and those that do not have the mutation (2) the impact of 5-FU , the most common colorectal cancer drug on colorectal cancer cells that have EGFR mutation and those that do not have the mutation according to the concentration time.

The setting of the experiment is determined after reading several similar studies.

		1	2	3	4	5	6
PLA2	1500u/ml	0	1	3	5	7	10	u/ml
		0	0.33	0.99	1.65	2.31	3.33	Ul
5-FU	10mg/ml	0	100	200	300	400	500	uM
		0	0.65	1.3	1.95	2.6	3.25	ul

(chart. 1)

Approximate final volume: 500ul

I planned to examine the result after 72 hours later.

 

Method:

1.

RPMI 1640= glucose + buffering chemical + PH indicator+ FBS + PS

TE buffer

1.     Suck RPMI 1640.

2.     Put TE buffer and wait 7 minutes (5-10minutes)

TE buffer is for getting cells that were originally adjoined in the floor of plate.

(cells should not be with TE buffer more than 10 minutes because cell can get damage.

3.     Separate cells and TE buffer using centrifuge

4.     Suck TE butter

Leave a little amount of buffer. (Cells can be sucked together if I try to suck entire buffer)

5.     Put RPMI 1640

6.     Put 500ul of solution (RPMI 1650 + colorectal cell) in small plates.

7.     Put PLA2 and 5 FU (chart. 1)

8.     Store the completed plated in the culture medium