"
Cathy Liu's notebook
From 2009.igem.org
08.14-09.11
Continued to screen receptors and constructs via Transwell Assay.
08.13
I. Transfections
Car1-FRB: 2.6ul DNA
hM4: 2.5ul DNA
SSF-YFP-hM4D-βPix: 2.1ul DNA
hM4D Act A Long: 2.45ul DNA
hM4D Act A Short: 2.1ul DNA
hM4D LPD Short: 2.6ul DNA
pMXS-Puro: .32ul DNA
II. Transwell Assay
Chemoattractant Dilutions:
CNO [0nM, 10nM, 100nM, 1uM]
cAMP [0nM, 10nM, 100nM, 1uM]
fMLP [100nM]
Notes:
Transfections: very low cell count
Transwells: low cell count & lost input cells for Act A Short and LPA Short
Only hM4 showed response.
08.12
I. Transfections
CCR7 - Not enough ligand
GPR132: 1.85ul DNA
LPA1: 1.8ul DNA
OPRL1: 2.1ul DNA
pMXS-Puro: .32ul DNA
II. Transwells
Chemoattractant Dilutions:
LPC: [0nM, 10nM, 100nM, 1uM]
LPA: [0nM, 100nM, 500nM, 1uM]
Orphanin FQ: [0nM, 1nM, 10nM, 100nM]
fMLP [100nM]
96-well plate
A1-12: GPR132 (0nM, 10nM, 100nM, 1uM)
B1-12: LPA1 (0nM, 100nM, 500nM, 1uM)
C1-12: OPRL1 (0nM, 1nM, 10nM, 100nM)
D1-3: GPR132 fMLP
D4-6: LPA1 fMLP
D7-9: OPRL1 fMLP
E1-3: GPR132 input
E4-6: LPA1 input
E7-9: OPRL1 input
E10-12: Media
F1-3: Transfection Efficiency
Only OPRL1 shows migration.
08.06
I. Transfections
ADRA1A: Epinephrine
EDG1: S1P
GPR132: not enough cells
GRM2: Glutamate
GRM4: Glutamate
LPA1: mistake
MTNR1A: Melatonin
OPRL1: Orphanin FQ
V1B: Vasopressin
II. Transwells
Chemoattractant Dilutions:
See 08.04 (no LPA or LPC)
96-well plate:
Plate 1
A1-12: ADRA1A
B1-12: EDG1
C1-12: GRM2
D1-12: GRM4
E1-12: MTNR1A
F1-12: OPRL1
G1-12: VIB
Plate 2 A1-3: ADRA1A fMLP
A4-6: EDG1 fMLP
A7-9: GRM2 fMLP
A10-12: GRM4 fMLP
B1-3: MTNR1A fMLP
B4-6: OPRL1 fMLP
B7-9: V1B fMLP
C1-3: ADRA1A Input
C4-6: EDG1 Input
C7-9: Grm2 Input
C10-12: MTNR1A Input
D1-3: OPRL1 Input
D4-6: V1B Input
D7-9: Media
D10-12: Grm4 Input
08.05
I. Transfections:
pMXS-Puro (WT)/GPCR+ pMAXGFP
CCR7: MIP-3Beta
DOR: DADLE
DOR-ERM: DADLE
DOR-EZRIN: DADLE
DOR-KIFC: DADLE
DOR-VHEAD: DADLE
hM3: CNO
hM3.2: CNO
II. Transwell
8 GPCRs - 8 GPCR Plates
2 fMLP plates; 8 wells for input
Chemoattractant Dilutions:
MIP-3Beta[0ug/ml, 0.01ug/ml, 0.1ug/ml, 1ug/ml]
DADLE [0nM, 10nM, 100nM, 1uM]
CNO [0nM, 10nM, 100nM, 1uM]
96-well plate:
Plate 1
A1-12: DOR KIFC
B1-12: DOR
C1-12: DOR ERM
D1-12: DOR EZRIN
E1-12: CCR7
F1-12: DOR VHEAD
G1-12: hM3
H1-12: hM3.2
Plate 2
A1-3: CCR7 fMLP
A4-6: DOR fMLP
A7-9: DOR ERM fMLP
A10-12: DOR EZRIN fMLP
B1-3: DOR KIFC fMLP
B4: empty
B5-6: DOR VHEAD fMLP
B7-9: hM3
B10-12: hM3.2
C1-3: CCR7 Input
C4-6: DOR input
C7-9: DOR ERM input
C10-12: DOR EZRIN input
D1-3: DOR KIFC input
D4-6: DOR VHEAD
D7-9: hM3
D10-12: hM3.2
E1-9 Transfection cells (one well per gpcr)
E10-12: Media
F1: DOR VHEAD fMLP
Wildtype cells did not stain. Redo GPCRs and RASSLs.
08.04
I. Transfections
ADRA1A - Epinephrine
CCR7 not enough cells
EDG1 - S1P
GPR132 - LPC
GRM2 - Glutamate
GRM4 - Glutamate
hM3 - CNO
LPA1 - LPA
MTNR1A - Melatonin
OPRL1 - Orphanin FQ
V1B - Vaspressin
pMXS-Puro (WT)
II. Transwell Assay
Chemoattractant Dilutions:
Epinephrine: [0nM, 10nM, 100nM, 1000nM]
S1P: [0nM, 10nM, 100nM, 1uM]
LPC: [0nM, 10nM, 100nM, 1uM]
Glutamate: [0nM, 10nM, 100nM, 1uM]
CNO [0nM, 10nM, 100nM, 1uM]
LPA: [0nM, 100nM, 500nM, 1uM]
Melatonin: [0nM, 1nM, 10nM, 1uM]
Orphanin FQ: [0nM, 1nM, 10nM, 100nM]
Vasopressin [0nM, 10nM, 100nM, 1uM]
fMLP: [100nM]
REDO SCREEN DUE TO GUAVA MALFUNCTION.
08.03
I. Transfections
1. AGTR1: 2.1uL*
2. AGTR2: 3uL
3. B2AR: 1.9uL
4. B2AR Ezrin: 2.8uL
5. GRM2 not enough cells
6. GRM4 has high toxicity so cells dont fluoresce*
7. hM2D plasmid concentration too low
8. hM3 not enough cells
9. hM3.2: 2.8uL
10. hM4: 2.5uL
11. HTR1A: 2uL
12. HTR2B: 2.8uL
13. HTR7A: 1.9uL
14. Rs1: 3uL
15. Rs1.3: 2uL
16. pCDNA: 2uL
- Arc discharge
II. Transwell
• Control-HL-60 dyed with Vybrant Red
• New lot of BD Falcon inserts
Chemoattractant dilutions:
1. 10uM stock Angiotensin II
[0nM, 1nM, 10nM, 100nM]
2. 50mM stock Glutamate
[0nM, 10nM, 100nM, 1uM]
3. 100mM stock Seratonin
[0nM, 10nM, 100nM, 1000nM]
4. CNO
[0nM, 10nM, 100nM, 1uM]
5. 29.4mM stock Zacopride
[0nM, 10nM, 100nM, 1uM]
6. fMLP [100nM]
07.28
I. Transfections
hM4: 2uL
pMaxGFP: 2uL
II. Transwell
1. RPMI Media
2. mHBSS + 0.1% BSA
Inserts: 3 plates CNO + RPMI: Millipose, BD, or Corning
3 plates CNO + mHBSS: Millipore, BD, or Corning
1 plate fMLP + Media: Millipore, BD, or Corning
1 plate fMLP + mHBSS: Millipore, BD, or Corning
Chemoattractant dilutions: 1. CNO [0nM, 10nM, 100nM, 1uM]
2. CNO [0nM, 10nM, 100nM, 1uM]
3. fMLP [10mM]
4. fMLP [10mM]
96-well plate:
A1-12: Millipore/Media [0nM, 10nM, 100nM, 1uM]
B1-12: BD/Media [0nM, 10nM, 100nM, 1uM]
C1-12: Millipore/BSA [0nM, 10nM, 100nM, 1uM]
D1-12: BD/BSA [0nM, 10nM, 100nM, 1uM]
E1-3: Corning fMLP/Media
E4-6: Millipore fMLP/Media
E7-9: BD fMLP/Media
F1-3: Corning fMLP/BSA
F4-6: Millipore fMLP/BSA
F7-9: BD fMLP/BSA
F12: Corning/BSA 10nM
G1-12: Corning/Media [0nM, 10nM, 100nM, 1uM]
H1-12: Corning/BSA [0nM, 10nM, 100nM, 1uM]
- All fMLP is 10nM
- H4 is wrong, H4's data is F12
BD Falcon remains insert of choice.
07.21
I. Transfections
1. ADRA1A: 1.8uL
2. B2AR: 1.9uL
3. B2AR-Ezrin: 2.5uL
4. HTR1A: 2uL
5. HTR2B: 2.8uL
6. GPR132: 1.5uL
7. LPA1: 1.8uL
8. pCDNA: 2.1uL
9. pMaxGFP: 2uL
II. Transwell
Chemoattractant dilutions:
1. 100mM stock Epinephrine Hydrochloride
[0nM, 10nM, 100nM, 1uM]
2. 100mM stock Isoproterenol Hydrochloride (Isoprenaline)
[0nM, 100pM, 1nM, 10nM]
3. 5mM stock Lysophosphatidic Acid (LPA)
[0nM, 100nM, 500nM, 1uM]
4. 25mM stock Lysophosphatidyl Choline (LPC)
[0nM, 10nM, 100nM, 1uM]
5. 100mM stock Seratonin
[0nM, 10nM, 100nM, 1000nM]
- Not enough cells for desired count: lowered
III. Analysis of 07.20 Transwell
07.20
I. Transfections
1. AGTR1
2. AGTR2: 2.9uL
3. CCR7: 2.5uL
4. GRM2: 1.9uL
5. GRM4: 2.3uL
6. MTNR1A: 2.1uL
7. OPRL1: 2.1uL
8. V1B: 2.4uL
9. pCDNA: 2.1uL
10. pMaxGFP: 2uL
II. Transwell
1. 50mM stock Glutamate
[0nM, 10nM, 100nM, 1uM]
2. 500uM stock Orphanin FQ
[0nM, 1nM, 10nM, 100nM]
3. 100mM stock Melatonin
[0nM, 1nM, 10nM, 1uM]
4. 10uM stock Angiotensin II
[0nM, 1nM, 10nM, 100nM]
5. 100ug/mL stock MIP-3Beta
[0ug/mL, 0.01ug/mL, 0.1ug/mL, 1ug/mL]
6. 5mM stock Vasopressin
[0nM, 10nM, 100nM, 1uM]
96-well plates:
Plate #1 (Triplicates)
A1-12: AGTR1 0nM, 1nM, 10nM, 100nM
B1-12: AGTR2 0nM, 1nM, 10nM, 100nM
C1-12: CCR7 0nM, 0.01nM, 0.1nM, 1nM
D1-12: GRM2 0nM, 10nM, 100nM, 1uM
E1-12: GRM4 0nM, 10nM, 100nM, 1uM
F1-12: MTNR1A 0nM, 1nM, 10nM, 1uM
G1-12: OPRL1 0nM, 1nM, 10nM, 100nM
H1-12: VIB 0nM, 10nM, 100nM, 1uM
Plate #2 (A-F Duplicates, G & H Triplicates)
A1-8: Angiontensin II 0nM, 1nM, 10nM, 100nM
B1-8: Mip-3Beta 0nM, 0.01nM, 0.1nM, 1nM
C1-8: Melatonin 0nM, 1nM, 10nM, 1uM
D1-8: Glutamate 0nM, 10nM, 100nM, 1uM
E1-8: Orphanin FQ 0nM, 1nM, 10nM, 100nM
F1-8: Vasopressin 0nM, 10nM, 100nM, 1uM
G1-3: ATGR1/fMLP
G4-6: AGTR2/fMLP
G7-9: GRM2/fMLP
G10-12: CCR7/fMLP
H1-3: GRM4/fMLP
H4-6: MTNR1A/fMLP
H7-9: OPRL1/fMLP
H10-12: VIB/fMLP
Plate #3
A1-3: fMLP WT
A4-6: Media
A7-9: Input WT
B1-3: Input AGTR1
B4-6: Input AGTR2
B7-9: Input CCR7
B10-12: GRM2
C1-3: Input GRM4
C4-6: Input MTNR1A
C7-9: Input OPRL1
C10-12: Input VIB
07.15
I. Transfections
1. AGTR1: 2uL
2. AGTR2: 3uL
3. GRM2: 2uL
4. GRM4: 2.3uL
5. hM4: 1uL
6. MTNR1A: 2uL
7. OPRL1: 2uL
8. pCDNA: 2uL
9. pMaxGFP: 2uL
8:40
5-hour incubation at 37C
II. Transwell
Chemoattractant dilutions: 1. 10nM stock Angiotensin II
[0nM, 10pM, 1nM, 10nM]
2. 500uM stock Orphanin FQ
[0nM, 1nM, 10nM, 100nM]
3. 50mM stock Glutamate
[0nM, 10nM, 100nM, 1uM]
4. 100mM stock Melatonin
[0nM, 1nM, 10nM, 1uM]
07.09
1. Minipreps (249.4 ng/uL)
2. Digestion, run gel
3. FACs Analysis
07.08
I. Transfections: hM4 RASSLs
In 5-day-differntiated HL-60:
1. L1+pMaxGFP
2. L2+pMaxGFP
3. L3+pMaxGFP
4. M1+pMaxGFP
5. M2+pMaxGFP
6. H1+pMaxGFP
7. H2+pMaxGFP
8. H3+pMaxGFP
II. Transwell Assay
Chemoattractant dilution:
1. 10mM stock CNO
07.07
I. Amaxa Cotransfection
In 5-day-differentiated HL-60:
1. hM2+pMaxGFP
2. hM3+pMaxGFP
3. hM4+pMaxGFP
4. pCDNA3.1+pMaxGFP
- Unnecessary second spin for samples
4-hour incubation at 37C
II. Transwell Assay
12-well plates: CNO (light sensitive)
Control plate: 10nM fMLP
Chemoattractant dilutions:
1. 10mM stock CNO
2. 10mM stock fMLP
Cell dilutions: 100,000cells/400uL (8mL)
1. pCDNA
2.65mL cells + 5.35mL Media
2. hM2
4.3mL cells + 3.7mL Media
3. hM3
3.88mL cells + 4.12mL Media
4. hM4
3.85mL cells + 4.15mL Media
30-minute incubation at 37C
Remove wells; add 12uL EDTA to each sample
Pipet samples into eppendorfs
Pipet 100uL into 96-well plate
+ 100uL fixation + 25uL beads
96-well plate
A: hM2 CNO
B: hM3 CNO
C: hM4 CNO
D: pCDNA CNO
E: hM2 fMLP, hM3 fMLP, hM4 fMLP, pCDNA fMLP
F: hM2 input cells, hM3 input cells, hM4 input cells
G: control input cells
07.06
I. Transfection: Amaxa for HL-60 cells
Materials
• 5 x 106 5-day-differentiated HL-60 per transfection
• 2.5mL IMDM media from Gibco per reaction
• Amaxa Nucleofactor Kit V. Premix solutions and date bottle (good for 2 months)
• 24-well cell culture plates
• 1.5mL eppendorfs
Protocol
1. Check cells under light microscope to make sure they look healthy (morphological polarity).
2. Set up post-transfection recovery plate by adding 1mL of IMDM media to a well in a 24-well plate (2 well/transfection). In a separate well at 0.5mL of IMDM media per transfection to an empty well. Equilibrate plate at 37°C in 5% CO2 for 20+ minutes.
3. Pipet 5 x 106 5-day-differentiated cells into a 15mL falcon tube (1tube/transfection). Spin down cells at 90g for 10 minutes.
4. During centrifuge spin, turn on Amaxa Nucleofactor Device and select program Y-001.
5. Prepare transfection reagents.
6. Pipet 500uL of equilibrated IMDM media from step #2 into eppendorf. Do one for each transfection. Place in cell culture incubator.
7. Aspirate media from cell pellet. Remove as much media as possible: extra media can interfere with effectiveness of electroporation and cells pellets exposed to air are easier to resuspend.
8. Pipet 100uL DNA and Nucleofactor solution and resuspend cells by flicking tube.
9. Pipet into electroporation cuvette (provided by Amaxa kit) and immediately zap in Nucleofactor. Remove eppendorf containing 500mL of IMDM from incubator.
10. Use pasture pipet (provided by Amaxa kit) to add 500uL warm IMDM media for eppendorfs prepared in step #6 and pipet back into labeled eppendorf. Let cells recover/settle/equilibrate at 37°C in 5% CO2 for 30-60 minutes (until cells settle). Letting cells settle allows them to recover at high cell concentrations (higher cell-cell contact leads to healthier cells), but leaving them into too long can be bad because tube does not allow for continuous equilibration with CO2.
11. Repeat steps #7-10 for each transfection.
12. When cells from step #10 have settled. Invert tube to resuspend them and pipet 260uL into two wells in prepared 24-well plates.
13. Let recover/express for 2-6 hours. Use FACs or microscope to check expression levels.
II. HL-60 Transfection Media
III. Minipreps
Alpha-Pix
B2AR
BPRX-GFP-Vasp
DOR-Erm
DOR-Ezrin
GFP-Vasp-BPRX
Intersectin
LPD 775-1250
P-Rex 5
IV. GPCR Organization
V. Transformations
ActA 30-612
ActA 225-392
B2AR Actinin
Beta-Pix
DOR-Actinin DOR-Kifc
LPD 775-1250
Vav
07.02
I. FACs
Beads: 1,000,000/mL → 25,000/25uL
II. FlowJo
P1: Live cell population
P2: Beads
P3: Live green cell population
Corrected live green cell population
- cells acquired/# beads acquire x 11,111 beads
Migration (%)
- cells acquired/average # input cells x 100
07.01
I. Transwell Assay
Protocol
1. Label 12-well plates.
2. Sigmacote 12-well plates.
3. Filter cells.
4. Count cells.
5. Centrifuge cells: 1000rpm for 5 minutes.
6. Dilute chemoattractants.
7. Aliquot 1mL/well.
8. Aspirate.
9. Resuspend with 5mL media.
10. Dilute cells: 250,000/mL. 250,000x7mL = 1,750,000 cell
11. Place inserts into each well.
12. Pipet 400uL cells into each insert.
13. Incubate: 37C, 5% CO2 for 30 minutes.
14. Pipet 12uL 0.5M EDTA into each well.
15. Tap plate, remove inserts.
16. Resuspend solution, transfer to eppendorf.
17. Transfer 100uL into 96-well plate.
25uL beads + 100uL Fixation Buffer + 100uL cells
or 25uL Media + 100uL Fixation Buffer + 100uL cells
II. 0.5M EDTA
06.29
I. Bare Transwell Assay with Flow Cytometry Count for HL-60 Chemotaxis
II. Flow Cytometry
III. HL-60 Media
IV. Chemoattractant dilutions
fLMP: 10nM stock solution
1uM → 100nM → 10nM → 0nM
V. Count cells
VI. 0.5M EDTA
