Ryan Liang's notebook

8/13 Tranfections

1. Car1-FRB

2. hM4

3. SSF-YFP-hM4D-βPix

4. hM4D Act A Long

5. hM4D Act A Short

6. hM4D LPD Short

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%

Chemoattractant Dilutions

CNO [0nM, 10nM, 100nM, 1uM]

cAMP [0nM, 10nM, 100nM, 1uM]

fMLP [100nM]

Result: hM4D worked only

8/12

Transfections

1. GPR132

2. LPA1

3. OPRL1

4. Control

Transfection Efficiency:

GPR132: 41%

LPA1: 43.2%

OPRL1: 50%

Chemoattractant Dilutions

LPC: [0nM, 10nM, 100nM, 1uM]

LPA: [0nM, 100nM, 500nM, 1uM]

Orphanin FQ: [0nM, 1nM, 10nM, 100nM]

fMLP [100nM]

RESULTS: OPRL1 worked only

8/10

Transfections

1. DOR

2. DOR ACTININ

3. DOR ERM

4. DOR EZRIN

5. DOR KIFC

6. DOR VHEAD

7. hM2D

8. hM3D

9. hM3.2D

10. Control

Transfection Efficiency:

DOR: 40.9%

DOR ACTININ: 39.5%

DOR ERM: 47.5%

DOR EZRIN: 48.1%

DOR KIFC: 39.2%

DOR VHEAD: 32.1%

hM2D: 35.2%

hM3D: 51%

hM3.2D: 54.7%

Chemoattractant Dilutions

DADLE [0nM, 1nM, 10nM, 100nM]

CNO [0nM, 10nM, 100nM, 1uM]

fMLP [100nM]

Results: DOR, DOR ACTININ, DOR EZRIN, DOR KIFC, DOR VHEAD, and hM3.2D worked

8/6

Transfections

1. ADRA1A

2. EDG1

3. GRM2

4. GRM4

5. MTNR1A

6. OPRL1

7. VIB

8. Control

Chemoattractant Dilutions

Epinephrine: [0nM, 10nM, 100nM, 1000nM]

S1P: [0nM, 10nM, 100nM, 1uM]

Glutamate: [0nM, 10nM, 100nM, 1uM]

CNO [0nM, 10nM, 100nM, 1uM]

Melatonin: [0nM, 1nM, 10nM, 1uM]

Orphanin FQ: [0nM, 1nM, 10nM, 100nM]

Vasopressin [0nM, 10nM, 100nM, 1uM]

fMLP: [100nM]

8/5

Transfections

1. CCR7

2. DOR

3. DOR ERM

4. DOR EZRIN

5. DOR KIFC

6. DOR VHEAD

7. hM3

8. Hm3.2

9. Control

Transfection Efficiency:

CCR7: 45.2%

DOR: 62.8%

DOR ERM: 45.7%

DOR EZRIN: 40%

DOR KIFC: 35.2%

DOR VHEAD: 42.4%

hM3: 47.4%

hM3.2: 30.9%

Chemoattractant Dilutions:

MIP-3Beta (0ug/ml, 0.01ug/ml, 0.1ug/ml, 1ug/ml)

DADLE [0nM, 10nM, 100nM, 1uM]

CNO [0nM, 10nM, 100nM, 1uM]

RESULTS: Wildtype cells did not stain so our data is unreliable so we have to redo all these GPCRs and RASSLs. Of the results, it shows that CCR7, DOR, DOR EZRIN, DOR KIFC, DOR VHEAD, and hM3.2 migrated. DOR KIFC is a maybe. When we redo it, we will change the concentrations to 10x lower.

8.4

Transfections

1.ADRA1A

2.EDG1

3.GPR132

4.GRM2

5.GRM4

6.hM3

7.LPA1

8.MTNR1A

9.OPRL1

10.V1B

11. Control

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]

RESULTS: None, machine broke, redo 8/6

8/3

Transfections

1. AGTR1

2. AGTR2

3. B2AR

4. B2AR EZRIN

5. hM3.2

6. hM4

7. HTR1A

8. HTR2B

9. HTR7A

10. Rs1

11. Rs1.3

12. Control

Chemoattractant Dilutions:

Isoprenaline [0nM, 1nM, 10nM, 100nM]

CNO: [0nM, 10nM, 100nM, 1uM]

Zacopride: [0nM, 10nM, 100nM, 1uM]

Angiontensin II (0nM, 1nM, 10nM, 100nM)

Seratonin (0nM, 10nM, 100nM, 1000nM)

Glutamate [0nM, 10nM, 100nM, 1uM]

fMLP: [100nM]

7/31

Goal: Combine WT cells and GPCR cells in the same well/insert only if we can give WT RFP.

1. Transfection with DsRed2 and mCherry

2. Dye cells with Red Vybrant @ 0,1,2,5,15,20 minutes

DsRed2 and mCherry takes too long to show RFP (5+ hours)

Cells are dyed at the optimum amount by 15minutes.

7/29

Transwell

Test new and old stocks of CNO in Millipore and BD

Chemoattractant Dilutions:

fMLP [100nM]

CNO [10nM, 100nM] Old & New

7/28

Transfections

hM4 Transients in Millipore, BD, and Corning

Chemoattractant Dilutions:

CNO [0nM, 10nM, 100nM, 1uM]

fMLP [10mM]

RESULTS: BD is best

7/27

Site DIrected Mutagenesis of DOCK insert in pTOPO

Picked colonies for AB-SSF pDONR221-PL-Flag, PCR2.1 TOPO-ITSN, PCR2.1 TOPO-TUBA, pDONR221-LPD775-1250aa, AB-YFP pDONR221-PL-FLAG-YFP, and TOPO-LPD full

7/21

Transfections

1. ADRA1A

2. B2AR

3. B2AR EZRIN

4. HTR1A

5. HTR2B

6. GPR132

7. LPA1

8. Control

Chemoattractant Dilutions:

Epinephrine (0nM, 10nM, 100nM, 1000nM)

Isoprenaline(0nM, 100pM, 1nM, 10nM)

LPA (0nM, 100nM, 500nM, 1uM)

LPC (0nM, 10nM, 100nM, 1uM)

Serotonin (0nM, 10nM, 100nM, 1000nM)

7/20

Transfections

1. AGTR1

2. AGTR2

3. CCR7

4. GRM2

5. GRM4

6. MTNR1A

7. OPRL1

8. VIB

9. Control

Dilution Calculations

Angiontensin II (0nM, 1nM, 10nM, 100nM)

MIP-3Beta (0ug/ml, 0.01ug/ml, 0.1ug/ml, 1ug/ml)

Vasopressin (0nM, 10nM, 100nM, 1uM)

Orphanin FQ [0nM, 1nM, 10nM, 100nM]

Glutamate [0nM, 10nM, 100nM, 1uM]

Melatonin [0nM, 1nM, 10nM, 1uM]

7/15

Transfections

1. AGTR1

2. AGTR2

3. GRM2

4. GRM4

5. MTNR1A

6. OPRL1

7. Control

7/14

Redo Unsuccessful Control Digestions

SAMPLE	5' ENZYME 	 3' ENZYME	 BUFFER	 POSITIVE GRM4	 BamH1	             Xba1	 2	 YES HTR7A	 EcoR1	             Xba1	 2	 NO GPR132	 EcoR1	              Xho1	 2	 YES EDG1	 BamH1	             Xho1	 2	 NO RO2	 Xma1	              none	 4	 NO Rs1.3	 Not1	              none	 3	 NO hM4D	 Nhe1	              none	 2	 YES hM2D	 Xma1	             none	 4	 NO Rs1	 HindIII              none	 2	 YES

Dump unsuccessful control samples, either we retransform or repick colonies.

7/13

Miniprep/Aar1 digestion of hM4D, Rs1.3, YFP, SSF

Digestion#2 (CONTROL DIGEST) PLASMID	5' ENZYME 	3' ENZYME 	INSERT SIZE 	BUFFER 	 POSITIVE MNTR1A            EcoR1	    Xho1	      1052	 2	 YES GRM4	           BamH1	    Xba1	      2739	 2	 NO AGTR2	            EcoR1	    Xho1	      1092	 2	 YES AGTR1	           EcoR1	    Xho1	      1080	 2	 YES GRM2	           BamH1	    Xho1	      2619	 3	 YES HTR7A	           EcoR1	    Xba1	      1338	 2	 NO HTR2B	            BamH1           Xho1	      1445	 3	 YES V1B	           BamH1   	    Xho1	      1275	 3	 YES CCR7	           EcoR1	    Xho1	      1137	 2	 YES GPR132	           EcoR1	    Xho1	      1143	 2	 NO EDG1	            BamH1	    Xho1	      1149	 3	 NO EDG2	            BamH1	    Xho1	      1095	 3	 YES OPRL1	           EcoR1	    Xho1	      1113	 2	 YES ADRA1A	           EcoR1	    Xho1	      1401	 2	 YES HTR1A	           BamH1	    Xho1	      1270	 3	 YES Ro2	           Xma1	    none              1646	 4	 NO hM3.2	            BamH1	    none	      944	 3	 YES Rs1.3	           Not1	    none	      1448	 3	 NO hM2D	            Xma1	    none	      1353	 4	 NO hM4D	            Nhe1	    none	      1260	 2	 NO

7/9 Did minipreps for multiple pMAX-GFP colonies, combined all the plasmids into one microcentrifuge and the yield was 249.4ng/ul.

Made a digestion, Cathy ran a gel

Cathy, and I worked on analyzing FACS results from Tuesday and Wednesday's transwells/transfections. Analyzed data on the Flowjo program; organized the data on excel, then put the results on a bar graph.

7/8 Transwell 8 variations of the RASSL hM4. The 8 were L1, L2, L3, M1, M2, H1, H2, and H3

Chemoattractant Dilutions CNO (0nM, 10nM, 100nM, 1uM)

7/7 Transfection (Contransfection) Add two constructs/vectors into our HL-60 cell line (RASSL + pMAXGFP) Use 5 days HL-60 differentiated with DMSO

Transfected hM2, hM3, hM4, Rs1.3, all including pMAXGFP into HL-60 cells Incubate in 37C for 4 hours. Used for transwell assay.

CNO Dilution is 10nM fMLP Dilution is 10nM

7/6 We used Amaxa transfection to get pMAX GFP DNA into 4 days HL-60 cells

Protocol provided from: http://cellpropulsionlab.pbworks.com (copied over from limwiki.ucsf.edu)

100ml monocyte media for HL-60 cells

78ml Gibco IMDM

20ml 20% FBS

1ml Glutamate

1ml Antibiotic/Antimycotics

2.75ml Nucleofector solution

2.25ml Cell Line Nucleofector Solution V

.50ml Supplement

Positive transfection showed cells with green fluorescence, there was about 40% fluorescence meaning 40% success rate. We have to note that using the right centrifuge makes a difference in transfection results. Using the incorrect centrifuge resulted in lower cell count and a 30% fluorescence while the correct centrifuge resulted in a much higher cell count as well as a fluorescence of 70%.

GPCR Miniprep alpha-pix, intersectin, P-Rex 5, DOR-ERM, DOR EZRIN, B2AR, LPD 775-1250, BPRX GFP-VASP, GFP-VASP BPRX

Transformations DOR-KIFC, DOR-ACTININ, B2AR ACTININ, LPD 775-1250, ActA 30-612, ActA 225-392, Vav, and Beta-Pix

50ul  Competent cells (DH5-alpha)

50ul  1x PCM (40ul water/10ul 5x PCM)

1ul   Plasmid

101ul   TOTAL

Plated on CarB+Amp, incubating at 37C

7/2 Cathy, Caitlin and I learned how to analyze our cells with the FACS machine. Sometimes the FACS machine is not accurate, so we add beads to increase accuracy. The machine takes about 40 seconds per sample, we had 56 samples.

Beads stock is 1,000,000beads/ml, we used 25ul which is 25,000 beads. If we have 100 cells and 20,000 beads shown on the FACS, then we can assume that only 80% (20,000 of 25,000) of the cells and beads are shown, So 100% of the cells would be 125 cells. I learned how to analyze these numbers on a computer program Flowjo, excel, as well as on a graph.

7/1 Practice a second protocol runthrough of Transwell assay

6/29 First protocol runthrough of Transwell assay

6/25 Ran gels and checked dictyostellium confluency

6/23-6/18 Plate dicty, run gels, do minipreps, and purify plasmids