Hansi Liu's notebook

From 2009.igem.org

8.21

Dor-Actinin 

1. 7hr_DOR-act_1=0_2=1nM_3=10nM_4=100nM_5=fMLP_6=100nMfMLP
2. 8.5hr_DOR-act_1=0_2=1nM_3=10nM_4=100nM_5=1uM_6=100nMfMLP

Empty vector with pMAX 

1. 5hr_empty_1=0_2=1uMCNO_3=fMLP_hM4_4=0_5=1uMCNO_6=100nMfMLP
2. empty_12=0_34=1uMCNO_56=100nMfMLP

hM4 

1. 6.5hr_hM4_1=0_2=100nM_3=500nM_4=1uM_5=2uM_6=100nMfMLP

OPRL1 

1. OPRL1_7.5hr_1=0_2=10nM_3=100nM_4=1uM_5=fMLP_6=100nMfMLP
2. OPRL1_8.5hr_1=0_2=10nM_3=100nM_4=1uM_5=1uM_6=100nMfMLP

8.19

Dor-Actinin 

1. DOR-Act_10hr_1=0_2=1nM_3=10nM_4=100nMfMLP_5=100nM_6=100nMfMLP
2. DOR-Act_11h_1=0_2=1nM_3=10nM_4=10nM_5=100nM_6=100nMfMLP
3. DOR-Act_13h_1=0_2=1nM_3=10nM_4=10nM_5=100nM_6=100nMfMLP

8.13 hM4 pooled Stable cell(before sorting) 

1. 6uMchip_pool_1=0_2=50nMCNO_3=100nMCNO_4=0_5=1uMCNO_6=100fMLP
2. hM4pool_1=0_2=50nMCNO_3=100nMCNO_4=1uMCNO_5=2uMCNO_6=5uMCNO
3. hM4pool_1=0_2=50nMCNO_3=100nMCNO_4=1uMCNO_5=5uMCNO_6=100nMfMLP
4. hM4pool_1=0_2=100nMCNO_3=500nMCNO_4=1uMCNO_5=2uMCNO_6=100nMfMLP
5. hM4pool_1=02=50nMCNO_3=100nMCNO_4=1uMCNO_5=2uMCNO_6=100nMfMLP


8.12

hM3.2 

1. hM3.2_10hr_1=0_2=100nMCNO_3=500nMCNO_4=1uMCNO_5=2uMCNO_6=100nMfMLP

hM4 : try longer recovery 

1. hM4_12.5hr_1=0_2=100nMCNO_3=500nMCNO_4=1uMCNO_5=2uMCNO_6=100nMfMLP

OPRL1 

1. Oprl1_a7hr_1=0_2=100nMOrph_3=500nMOrph_4=1uMOrph_5=2uMOrph_6=100nMfMLP
2. Oprl1_b8hr_1=0_2=100nMOrph_3=500nMOrph_4=1uMOrph_5=2uMOrph_6=100nMfMLP

8.11 New protocol for transient cells come out

hM3.2 

1. hM3.2_8hr_1wash_1=0_2=100nMCNO_3=500nMCNO_4=1uMCNO_5=2uMCNO_6=100nMfMLP
2. hM3.2_5.5hr_2wash_1=0_2=100nMCNO_3=1uMCNO_4=2uMCNO_5=5uMCNO_6=100nMfMLP

hM4 

1. hM4_4.5hr_2wash_1=0_2=100nMCNO_3=1uMCNO_4=2uMCNO_5=5uMCNO_6=100nMfMLP
2. hM4_6.5hr_1wash_1=0_2=100nMCNO_3=500nMCNO_4=1uMCNO_5=2uMCNO_6=100nMfMLP
3. hM4_7.5hr_b1wash_1=0_2=100nMCNO_3=500nMCNO_4=100nMfMLP_5=1uMCNO_6=2uMCNO

8.4-8.10 optimise the condition for transient cells.

8.3 hM4 Transient cells: try higher concentration of CNO 

1. hm4_1=0_2=50nMCNO_3=100nMCNO_4=1uMCNO_5=2uMCNO_6=5uMCNO
2. hm4_1=0_2=50nMCNO_3=100nMCNO_4=1uMCNO_5=2uMCNO_6=5uMCNO
3. hm4_1=0_2=50nMCNO_3=100nMCNO_4=1uMCNO_5=2uMCNO_6=5uMCNO
4. hm4_1=0_2=50nMCNO_3=100nMCNO_4=1uMCNO_5=100nMfMLP_6=2uMCNO


8.3 hM4 Transient cells: try higher concentration of CNO 

1. hm4_1=0_2=50nMCNO_3=100nMCNO_4=1uMCNO_5=2uMCNO_6=5uMCNO
2. hm4_1=0_2=50nMCNO_3=100nMCNO_4=1uMCNO_5=2uMCNO_6=5uMCNO
3. hm4_1=0_2=50nMCNO_3=100nMCNO_4=1uMCNO_5=2uMCNO_6=5uMCNO
4. hm4_1=0_2=50nMCNO_3=100nMCNO_4=1uMCNO_5=100nMfMLP_6=2uMCNO


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.
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