Hansi Liu's notebook

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7.31 FACS sorted hM4 stable cell line

hM4 High

1. starve for 1.5hr_CH1=16nMCNO_CH2=48nMCNO_CH3=100nMCNO_CH4=1uMCNO_CH5=1.5uMCNO_CH6=2uMCNO
2. starve for 1.5hr_CH1=16nMCNO_CH2=48nMCNO_CH3=100nMCNO_CH4=1uMCNO_CH5=1.5uMCNO_CH6=2uMCNO
3. starve for 1.5hr_CH12=0_CH34=1uMCNO_CH56=100nMfMLP

hM4 Low

1. CH1=16nMCNO_CH2=48nMCNO_CH3=100nMCNO_CH4=1uMCNO_CH5=1.5uMCNO_CH6=2uMCNO
2. CH1=16nMCNO_CH2=48nMCNO_CH3=100nMCNO_CH4=1uMCNO_CH5=1.5uMCNO_CH6=2uMCNO
3. CH1=16nMCNO_CH2=48nMCNO_CH3=100nMCNO_CH4=1uMCNO_CH5=1.5uMCNO_CH6=2uMCNO
4. CH23=0_CH456=100nMfMLP

7.28 Transient Cells

1. Empty vector+pMAX
   CH12=0_CH34=1uMCNO_CH56=100fMLP

2. pBR64(hM4)+pMAX
   CH1=0_CH2=50nMCNO_CH3=100nMCNO_CH4=1uMCNO_CH5=2uMCNO_6=5uMCNO
   CH1=0_CH2=50nMCNO_CH3=100nMCNO_CH4=1uMCNO_CH5=2uMCNO_6=5uMCNO

7.27 FACS sorted hM4 stable cell lines

hm4High
1. CH12=0_CH34=100nMCNO_CH56=100nMfMLP
2. CH1=0_CH2=16nMCNO_CH3=16nMCNO_CH4=1uMCNO_CH5=5uMCNO_CH6=5uMCNO
3. CH1=0_CH2=16nMCNO_CH3=48nMCNO_CH4=100nMCNO_CH5=1uMCNO_CH6=2uMCNO
4. CH1=0_CH2=16nMCNO_CH3=48nMCNO_CH4=100nMCNO_CH5=1uMCNO_CH6=1.5uMCNO
5. CH1=0_CH2=16nMCNO_CH3=48nMCNO_CH4=100nMCNO_CH5=1uMCNO_CH6=1.5uMCNO
6. CH1=0_CH2=48nMCNO_CH3=100nMCNO_CH4=1uMCNO_CH5=1.5uMCNO_CH6=2uMCNO
7. CH1=0_CH23=16nMCNO_CH4=1uMCNO_CH56=5uMCNO

7.22 Move on to Transient cells!

0. Before every experiment with transient Cells, we need to first prepare them: co-transfect different engineered plasmids with pMAX-GFP into wild type cells. Cells will then be recovered for 5 hours before any microscopy.

1. Empty vector+pMAX

   CH12=0_CH34=100nMfMLP_CH56=100nMCNO

2. pBR64(hM4)+pMAX

   CH1=0_CH2=50nMCNO_CH3=100nMCNO_CH4=500nMCNO_CH5=1uMCNO_CH6=1.5uMCNO
   CH1=0_CH2=50nMCNO_CH3=100nMCNO_CH4=500nMCNO_CH5=1uMCNO_CH6=1.5uMCNO
   CH1=0_CH2=50nMCNO_CH3=100nMCNO_CH4=1uMCNO_CH5=1.5uMCNO_CH6=100fMLP

Results are not good. Need to optimise the protocol for transient cells.

7.21 WT HL-60

1. CH12=0to0_CH34_0to4nMfMLP_CH56=0to8nMfMLP
2. CH12=0to32nMfMLP_CH34=0to64nMfMLP_CH56=0to80nMfMLP
3. CH1=110nMfMLP_CH2=120nMfMLP_CH3=130nMfMLP_CH4=140nMfMLP_CH5=150nMfMLP_CH6=0

7.20 WT HL-60

CH123=0to0_CH456=0to100nMCNO
Transient Cells
pBR64(hM4)+pMAX
CH1=0_CH2=0to16nMCNO_CH3=0to100nMCNO_CH4=0to160nMCNO_CH5=0to1uMCNO_CH6=0to100nMfMLP

7.17 EZ-Taxiscanning:

WT HL-60

1. CH23=0to0_CH456=0to100nMCNO
2. CH123=0to0_CH456=0to100nMfMLP

hM4 Stable Cell Line
1. High expression level
   CH1=0to0_CH2=0to16nMCNO_CH3=0to48nMCNO_CH4=0to100nMCNO_CH5=0to160nMCNO_CH6=0to1uMCNO
2. Medium expression level
   CH1=0to0_CH2=0to16nMCNO_CH3=0to48nMCNO_CH4=0to100nMCNO_CH5=0to160nMCNO_CH6=0to1uMCNO
3. Low expression level
   CH1=0to0_CH2=0to16nMCNO_CH3=0to48nMCNO_CH4=0to100nMCNO_CH5=0to160nMCNO_CH6=0to1uMCNO

7.14 EZ-Taxiscanning:

WT HL-60
1. CH123=0to0nMfMLP_CH456=0to100nMfMLP_starve1.5hr
2. CH123=0to0nMfMLP_CH456=0to100nMfMLP_6um chip_starve1hr
3. CH12=0to16nMfMLP_CH34=0to48nMfMLP_CH56=0to160nMfMLP_starve1.5hr

hM4 Stable Cell Line
CH1=0to0nMCNO_CH2=100pMCNO_CH3=1nMCNO_CH4=10nMCNO_CH5=50nMCNO_CH6=100nMCNO_5uMchip

With the optimised protocol, after run the movies on mablab, we are able to get useful data about the characteristics of the movement of cells: speed, straightness, directionality, distance, etc.

7.3--7.13 Optimise the protocol

7.2

11:00
Ready to get an ID card !

13:30-14:30
Angi taught us how to analysis scan result(cell tracking) using a cool Matlab program written by herself.

15:00-
Completed the safety training courses on-line. Ready to do experiments~

later:
Mini Prep: 8 samples from Eric.