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Cathy Liu's notebook
From 2009.igem.org
--CathyLiu 03:39, 22 October 2009 (UTC)--CathyLiu 03:39, 22 October 2009 (UTC)[[ == 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
Transfection Efficiency
Car1-FRB: 42.4% hM4: 42.4% SSF-YFP-hM4D-βPix: 40.4% hM4D Act A Long: 46.4% hM4D Act A Short: 36.5% hM4D LPD Short: 44.3%
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] 1ul 10mM stock + 999ul media = 1ml 10uM 500ul 10uM + 49.5ml media = 50ml 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)
0nM 10nM 100nM 1uM 0nM 10nM 100nM 1uM 0nM 10nM 100nM 1uM
Control plate: 10nM fMLP
hM2 hM3 hM4 pCDNA hM2 hM3 hM4 pCDNA hM2 hM3 hM4 pCDNA
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
