Cathy Liu's notebook

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


 * 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