Cathy Liu's notebook

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--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

  1. cells acquired/# beads acquire x 11,111 beads

Migration (%)

  1. 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