Hansi Liu's notebook

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.