Cathy Liu's notebook

From 2009.igem.org

(Difference between revisions)
(New page: --~~~~--~~~~[[ == '''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-β...)
 
Line 1: Line 1:
-
--[[User:CathyLiu|CathyLiu]] 03:39, 22 October 2009 (UTC)--[[User:CathyLiu|CathyLiu]] 03:39, 22 October 2009 (UTC)[[
+
'''08.14-09.11'''
-
== '''08.14-09.11'''
+
Continued to screen receptors and constructs via Transwell Assay.
Continued to screen receptors and constructs via Transwell Assay.
Line 9: Line 8:
Car1-FRB: 2.6ul DNA
Car1-FRB: 2.6ul DNA
 +
hM4: 2.5ul DNA
hM4: 2.5ul DNA
 +
SSF-YFP-hM4D-βPix: 2.1ul DNA
SSF-YFP-hM4D-βPix: 2.1ul DNA
 +
hM4D Act A Long: 2.45ul DNA
hM4D Act A Long: 2.45ul DNA
 +
hM4D Act A Short: 2.1ul DNA
hM4D Act A Short: 2.1ul DNA
 +
hM4D LPD Short: 2.6ul DNA
hM4D LPD Short: 2.6ul DNA
-
pMXS-Puro:  .32ul DNA
 
-
Transfection Efficiency
+
pMXS-Puro:  .32ul DNA
-
 
+
-
Car1-FRB: 42.4%
+
-
hM442.4%
+
-
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
II.    Transwell Assay
Line 36: Line 32:
Notes:
Notes:
 +
Transfections: very low cell count
Transfections: very low cell count
 +
Transwells: low cell count & lost input cells for Act A Short and LPA Short
Transwells: low cell count & lost input cells for Act A Short and LPA Short
Line 44: Line 42:
I.    Transfections
I.    Transfections
 +
CCR7 - Not enough ligand
CCR7 - Not enough ligand
 +
GPR132: 1.85ul DNA
GPR132: 1.85ul DNA
 +
LPA1: 1.8ul DNA
LPA1: 1.8ul DNA
 +
OPRL1: 2.1ul DNA
OPRL1: 2.1ul DNA
 +
pMXS-Puro: .32ul DNA
pMXS-Puro: .32ul DNA
Line 63: Line 66:
96-well plate
96-well plate
 +
A1-12: GPR132 (0nM, 10nM, 100nM, 1uM)
A1-12: GPR132 (0nM, 10nM, 100nM, 1uM)
 +
B1-12: LPA1 (0nM, 100nM, 500nM, 1uM)
B1-12: LPA1 (0nM, 100nM, 500nM, 1uM)
 +
C1-12: OPRL1 (0nM, 1nM, 10nM, 100nM)
C1-12: OPRL1 (0nM, 1nM, 10nM, 100nM)
 +
D1-3: GPR132 fMLP
D1-3: GPR132 fMLP
 +
D4-6: LPA1 fMLP
D4-6: LPA1 fMLP
 +
D7-9: OPRL1 fMLP
D7-9: OPRL1 fMLP
 +
E1-3: GPR132 input
E1-3: GPR132 input
 +
E4-6: LPA1 input
E4-6: LPA1 input
 +
E7-9: OPRL1 input
E7-9: OPRL1 input
 +
E10-12: Media
E10-12: Media
 +
F1-3: Transfection Efficiency
F1-3: Transfection Efficiency
Line 80: Line 94:
I.    Transfections
I.    Transfections
 +
ADRA1A: Epinephrine
ADRA1A: Epinephrine
 +
EDG1: S1P
EDG1: S1P
 +
GPR132: not enough cells
GPR132: not enough cells
 +
GRM2: Glutamate
GRM2: Glutamate
 +
GRM4: Glutamate
GRM4: Glutamate
 +
LPA1: mistake
LPA1: mistake
 +
MTNR1A: Melatonin
MTNR1A: Melatonin
 +
OPRL1: Orphanin FQ
OPRL1: Orphanin FQ
 +
V1B: Vasopressin
V1B: Vasopressin
Line 93: Line 116:
Chemoattractant Dilutions:
Chemoattractant Dilutions:
 +
See 08.04 (no LPA or LPC)
See 08.04 (no LPA or LPC)
96-well plate:
96-well plate:
 +
Plate 1
Plate 1
 +
A1-12: ADRA1A
A1-12: ADRA1A
 +
B1-12: EDG1
B1-12: EDG1
 +
C1-12: GRM2
C1-12: GRM2
 +
D1-12: GRM4
D1-12: GRM4
 +
E1-12: MTNR1A
E1-12: MTNR1A
 +
F1-12: OPRL1
F1-12: OPRL1
 +
G1-12: VIB
G1-12: VIB
Plate 2
Plate 2
A1-3: ADRA1A fMLP
A1-3: ADRA1A fMLP
 +
A4-6: EDG1 fMLP
A4-6: EDG1 fMLP
 +
A7-9: GRM2 fMLP
A7-9: GRM2 fMLP
 +
A10-12: GRM4 fMLP
A10-12: GRM4 fMLP
 +
B1-3: MTNR1A fMLP
B1-3: MTNR1A fMLP
 +
B4-6: OPRL1 fMLP
B4-6: OPRL1 fMLP
 +
B7-9: V1B fMLP
B7-9: V1B fMLP
 +
C1-3: ADRA1A Input
C1-3: ADRA1A Input
 +
C4-6: EDG1 Input
C4-6: EDG1 Input
 +
C7-9: Grm2 Input
C7-9: Grm2 Input
 +
C10-12: MTNR1A Input
C10-12: MTNR1A Input
 +
D1-3: OPRL1 Input
D1-3: OPRL1 Input
 +
D4-6: V1B Input
D4-6: V1B Input
 +
D7-9: Media
D7-9: Media
 +
D10-12: Grm4 Input
D10-12: Grm4 Input
Line 125: Line 171:
I.    Transfections:  
I.    Transfections:  
 +
pMXS-Puro (WT)/GPCR+ pMAXGFP
pMXS-Puro (WT)/GPCR+ pMAXGFP
 +
CCR7: MIP-3Beta  
CCR7: MIP-3Beta  
 +
DOR: DADLE
DOR: DADLE
 +
DOR-ERM: DADLE
DOR-ERM: DADLE
 +
DOR-EZRIN: DADLE
DOR-EZRIN: DADLE
 +
DOR-KIFC: DADLE
DOR-KIFC: DADLE
 +
DOR-VHEAD: DADLE
DOR-VHEAD: DADLE
 +
hM3: CNO
hM3: CNO
 +
hM3.2: CNO
hM3.2: CNO
II. Transwell
II. Transwell
 +
8 GPCRs - 8 GPCR Plates
8 GPCRs - 8 GPCR Plates
 +
2 fMLP plates; 8 wells for input
2 fMLP plates; 8 wells for input
Chemoattractant Dilutions:
Chemoattractant Dilutions:
-
MIP-3Beta
+
MIP-3Beta[0ug/ml, 0.01ug/ml, 0.1ug/ml, 1ug/ml]
-
[0ug/ml, 0.01ug/ml, 0.1ug/ml, 1ug/ml]
+
DADLE [0nM, 10nM, 100nM, 1uM]
DADLE [0nM, 10nM, 100nM, 1uM]
Line 149: Line 205:
96-well plate:
96-well plate:
 +
Plate 1
Plate 1
 +
A1-12: DOR KIFC
A1-12: DOR KIFC
 +
B1-12: DOR
B1-12: DOR
 +
C1-12: DOR ERM
C1-12: DOR ERM
 +
D1-12: DOR EZRIN
D1-12: DOR EZRIN
 +
E1-12: CCR7
E1-12: CCR7
 +
F1-12: DOR VHEAD
F1-12: DOR VHEAD
 +
G1-12: hM3
G1-12: hM3
 +
H1-12: hM3.2
H1-12: hM3.2
Plate 2
Plate 2
 +
A1-3: CCR7 fMLP
A1-3: CCR7 fMLP
 +
A4-6: DOR fMLP
A4-6: DOR fMLP
 +
A7-9: DOR ERM fMLP
A7-9: DOR ERM fMLP
 +
A10-12: DOR EZRIN fMLP
A10-12: DOR EZRIN fMLP
 +
B1-3: DOR KIFC fMLP
B1-3: DOR KIFC fMLP
 +
B4: empty
B4: empty
 +
B5-6: DOR VHEAD fMLP
B5-6: DOR VHEAD fMLP
 +
B7-9: hM3
B7-9: hM3
 +
B10-12: hM3.2
B10-12: hM3.2
 +
C1-3: CCR7 Input
C1-3: CCR7 Input
 +
C4-6: DOR input
C4-6: DOR input
 +
C7-9: DOR ERM input
C7-9: DOR ERM input
 +
C10-12: DOR EZRIN input
C10-12: DOR EZRIN input
 +
D1-3: DOR KIFC input
D1-3: DOR KIFC input
 +
D4-6: DOR VHEAD
D4-6: DOR VHEAD
 +
D7-9: hM3
D7-9: hM3
 +
D10-12: hM3.2
D10-12: hM3.2
 +
E1-9 Transfection cells (one well per gpcr)
E1-9 Transfection cells (one well per gpcr)
 +
E10-12: Media
E10-12: Media
 +
F1: DOR VHEAD fMLP
F1: DOR VHEAD fMLP
Line 186: Line 271:
I.    Transfections
I.    Transfections
 +
ADRA1A - Epinephrine
ADRA1A - Epinephrine
 +
CCR7 not enough cells
CCR7 not enough cells
 +
EDG1 - S1P
EDG1 - S1P
 +
GPR132 - LPC
GPR132 - LPC
 +
GRM2 - Glutamate
GRM2 - Glutamate
 +
GRM4 - Glutamate
GRM4 - Glutamate
 +
hM3 - CNO
hM3 - CNO
 +
LPA1 - LPA
LPA1 - LPA
 +
MTNR1A - Melatonin
MTNR1A - Melatonin
 +
OPRL1 - Orphanin FQ
OPRL1 - Orphanin FQ
 +
V1B - Vaspressin
V1B - Vaspressin
 +
pMXS-Puro (WT)
pMXS-Puro (WT)
Line 220: Line 317:
Vasopressin [0nM, 10nM, 100nM, 1uM]
Vasopressin [0nM, 10nM, 100nM, 1uM]
 +
fMLP: [100nM]
fMLP: [100nM]
-
1ul 10mM stock + 999ul media = 1ml 10uM
 
-
500ul 10uM + 49.5ml media = 50ml 100nM
 
REDO SCREEN DUE TO GUAVA MALFUNCTION.
REDO SCREEN DUE TO GUAVA MALFUNCTION.
Line 231: Line 327:
1. AGTR1: 2.1uL*
1. AGTR1: 2.1uL*
 +
2. AGTR2: 3uL
2. AGTR2: 3uL
 +
3. B2AR: 1.9uL
3. B2AR: 1.9uL
 +
4. B2AR Ezrin: 2.8uL
4. B2AR Ezrin: 2.8uL
 +
5. GRM2 not enough cells
5. GRM2 not enough cells
 +
6. GRM4 has high toxicity so cells dont fluoresce*
6. GRM4 has high toxicity so cells dont fluoresce*
 +
7. hM2D plasmid concentration too low
7. hM2D plasmid concentration too low
 +
8. hM3 not enough cells
8. hM3 not enough cells
 +
9. hM3.2: 2.8uL
9. hM3.2: 2.8uL
 +
10. hM4: 2.5uL
10. hM4: 2.5uL
 +
11. HTR1A: 2uL
11. HTR1A: 2uL
 +
12. HTR2B: 2.8uL
12. HTR2B: 2.8uL
 +
13. HTR7A: 1.9uL
13. HTR7A: 1.9uL
 +
14. Rs1: 3uL
14. Rs1: 3uL
 +
15. Rs1.3: 2uL
15. Rs1.3: 2uL
 +
16. pCDNA: 2uL
16. pCDNA: 2uL
Line 252: Line 363:
•    Control-HL-60 dyed with Vybrant Red
•    Control-HL-60 dyed with Vybrant Red
 +
•    New lot of BD Falcon inserts
•    New lot of BD Falcon inserts
Line 257: Line 369:
1. 10uM stock Angiotensin II
1. 10uM stock Angiotensin II
 +
[0nM, 1nM, 10nM, 100nM]
[0nM, 1nM, 10nM, 100nM]
2. 50mM stock Glutamate
2. 50mM stock Glutamate
 +
[0nM, 10nM, 100nM, 1uM]
[0nM, 10nM, 100nM, 1uM]
3. 100mM stock Seratonin  
3. 100mM stock Seratonin  
 +
[0nM, 10nM, 100nM, 1000nM]
[0nM, 10nM, 100nM, 1000nM]
4. CNO
4. CNO
 +
[0nM, 10nM, 100nM, 1uM]
[0nM, 10nM, 100nM, 1uM]
5. 29.4mM stock Zacopride
5. 29.4mM stock Zacopride
 +
[0nM, 10nM, 100nM, 1uM]
[0nM, 10nM, 100nM, 1uM]
Line 278: Line 395:
hM4: 2uL
hM4: 2uL
 +
pMaxGFP: 2uL
pMaxGFP: 2uL
Line 283: Line 401:
1. RPMI Media
1. RPMI Media
 +
2. mHBSS + 0.1% BSA
2. mHBSS + 0.1% BSA
Inserts:
Inserts:
3 plates CNO + RPMI: Millipose, BD, or Corning
3 plates CNO + RPMI: Millipose, BD, or Corning
 +
3 plates CNO + mHBSS: Millipore, BD, or Corning
3 plates CNO + mHBSS: Millipore, BD, or Corning
 +
1 plate fMLP + Media: Millipore, BD, or Corning
1 plate fMLP + Media: Millipore, BD, or Corning
 +
1 plate fMLP + mHBSS: Millipore, BD, or Corning
1 plate fMLP + mHBSS: Millipore, BD, or Corning
Line 301: Line 423:
96-well plate:
96-well plate:
 +
A1-12: Millipore/Media [0nM, 10nM, 100nM, 1uM]
A1-12: Millipore/Media [0nM, 10nM, 100nM, 1uM]
 +
B1-12: BD/Media [0nM, 10nM, 100nM, 1uM]
B1-12: BD/Media [0nM, 10nM, 100nM, 1uM]
 +
C1-12: Millipore/BSA [0nM, 10nM, 100nM, 1uM]
C1-12: Millipore/BSA [0nM, 10nM, 100nM, 1uM]
 +
D1-12: BD/BSA [0nM, 10nM, 100nM, 1uM]
D1-12: BD/BSA [0nM, 10nM, 100nM, 1uM]
 +
E1-3: Corning fMLP/Media
E1-3: Corning fMLP/Media
 +
E4-6: Millipore fMLP/Media
E4-6: Millipore fMLP/Media
 +
E7-9: BD fMLP/Media
E7-9: BD fMLP/Media
 +
F1-3: Corning fMLP/BSA
F1-3: Corning fMLP/BSA
 +
F4-6: Millipore fMLP/BSA
F4-6: Millipore fMLP/BSA
 +
F7-9: BD fMLP/BSA
F7-9: BD fMLP/BSA
 +
F12: Corning/BSA 10nM
F12: Corning/BSA 10nM
 +
G1-12: Corning/Media [0nM, 10nM, 100nM, 1uM]
G1-12: Corning/Media [0nM, 10nM, 100nM, 1uM]
 +
H1-12: Corning/BSA [0nM, 10nM, 100nM, 1uM]
H1-12: Corning/BSA [0nM, 10nM, 100nM, 1uM]
*All fMLP is 10nM
*All fMLP is 10nM
 +
*H4 is wrong, H4's data is F12
*H4 is wrong, H4's data is F12
Line 325: Line 461:
1. ADRA1A: 1.8uL
1. ADRA1A: 1.8uL
 +
2. B2AR: 1.9uL  
2. B2AR: 1.9uL  
 +
3. B2AR-Ezrin: 2.5uL
3. B2AR-Ezrin: 2.5uL
 +
4. HTR1A: 2uL
4. HTR1A: 2uL
 +
5. HTR2B: 2.8uL
5. HTR2B: 2.8uL
 +
6. GPR132: 1.5uL
6. GPR132: 1.5uL
 +
7. LPA1: 1.8uL
7. LPA1: 1.8uL
 +
8. pCDNA: 2.1uL
8. pCDNA: 2.1uL
 +
9. pMaxGFP: 2uL
9. pMaxGFP: 2uL
Line 339: Line 483:
1. 100mM stock Epinephrine Hydrochloride
1. 100mM stock Epinephrine Hydrochloride
 +
[0nM, 10nM, 100nM, 1uM]
[0nM, 10nM, 100nM, 1uM]
2. 100mM stock Isoproterenol Hydrochloride (Isoprenaline)
2. 100mM stock Isoproterenol Hydrochloride (Isoprenaline)
 +
[0nM, 100pM, 1nM, 10nM]
[0nM, 100pM, 1nM, 10nM]
3. 5mM stock Lysophosphatidic Acid (LPA)  
3. 5mM stock Lysophosphatidic Acid (LPA)  
 +
[0nM, 100nM, 500nM, 1uM]
[0nM, 100nM, 500nM, 1uM]
4. 25mM stock Lysophosphatidyl Choline (LPC)  
4. 25mM stock Lysophosphatidyl Choline (LPC)  
 +
[0nM, 10nM, 100nM, 1uM]
[0nM, 10nM, 100nM, 1uM]
5. 100mM stock Seratonin  
5. 100mM stock Seratonin  
-
[0nM, 10nM, 100nM, 1000nM]
 
 +
[0nM, 10nM, 100nM, 1000nM]
*Not enough cells for desired count: lowered
*Not enough cells for desired count: lowered
Line 363: Line 511:
1. AGTR1
1. AGTR1
 +
2. AGTR2: 2.9uL
2. AGTR2: 2.9uL
 +
3. CCR7: 2.5uL
3. CCR7: 2.5uL
 +
4. GRM2: 1.9uL
4. GRM2: 1.9uL
 +
5. GRM4: 2.3uL  
5. GRM4: 2.3uL  
 +
6. MTNR1A: 2.1uL
6. MTNR1A: 2.1uL
 +
7. OPRL1: 2.1uL
7. OPRL1: 2.1uL
 +
8. V1B: 2.4uL
8. V1B: 2.4uL
 +
9. pCDNA: 2.1uL
9. pCDNA: 2.1uL
 +
10. pMaxGFP: 2uL
10. pMaxGFP: 2uL
Line 376: Line 533:
1.    50mM stock Glutamate
1.    50mM stock Glutamate
 +
[0nM, 10nM, 100nM, 1uM]
[0nM, 10nM, 100nM, 1uM]
2. 500uM stock Orphanin FQ
2. 500uM stock Orphanin FQ
 +
[0nM, 1nM, 10nM, 100nM]
[0nM, 1nM, 10nM, 100nM]
3. 100mM stock Melatonin
3. 100mM stock Melatonin
 +
[0nM, 1nM, 10nM, 1uM]
[0nM, 1nM, 10nM, 1uM]
  4. 10uM stock Angiotensin II
  4. 10uM stock Angiotensin II
 +
[0nM, 1nM, 10nM, 100nM]
[0nM, 1nM, 10nM, 100nM]
5. 100ug/mL stock MIP-3Beta
5. 100ug/mL stock MIP-3Beta
 +
[0ug/mL, 0.01ug/mL, 0.1ug/mL, 1ug/mL]
[0ug/mL, 0.01ug/mL, 0.1ug/mL, 1ug/mL]
6.  5mM stock Vasopressin
6.  5mM stock Vasopressin
 +
[0nM, 10nM, 100nM, 1uM]
[0nM, 10nM, 100nM, 1uM]
96-well plates:
96-well plates:
 +
Plate #1 (Triplicates)
Plate #1 (Triplicates)
 +
A1-12: AGTR1 0nM, 1nM, 10nM, 100nM
A1-12: AGTR1 0nM, 1nM, 10nM, 100nM
 +
B1-12: AGTR2 0nM, 1nM, 10nM, 100nM
B1-12: AGTR2 0nM, 1nM, 10nM, 100nM
 +
C1-12: CCR7  0nM, 0.01nM, 0.1nM, 1nM
C1-12: CCR7  0nM, 0.01nM, 0.1nM, 1nM
 +
D1-12: GRM2  0nM, 10nM, 100nM, 1uM
D1-12: GRM2  0nM, 10nM, 100nM, 1uM
 +
E1-12: GRM4  0nM, 10nM, 100nM, 1uM
E1-12: GRM4  0nM, 10nM, 100nM, 1uM
 +
F1-12: MTNR1A 0nM, 1nM, 10nM, 1uM
F1-12: MTNR1A 0nM, 1nM, 10nM, 1uM
 +
G1-12: OPRL1 0nM, 1nM, 10nM, 100nM
G1-12: OPRL1 0nM, 1nM, 10nM, 100nM
 +
H1-12: VIB      0nM, 10nM, 100nM, 1uM
H1-12: VIB      0nM, 10nM, 100nM, 1uM
 +
Plate #2 (A-F Duplicates, G & H Triplicates)
Plate #2 (A-F Duplicates, G & H Triplicates)
 +
A1-8: Angiontensin II 0nM, 1nM, 10nM, 100nM
A1-8: Angiontensin II 0nM, 1nM, 10nM, 100nM
 +
B1-8: Mip-3Beta 0nM, 0.01nM, 0.1nM, 1nM
B1-8: Mip-3Beta 0nM, 0.01nM, 0.1nM, 1nM
 +
C1-8: Melatonin 0nM, 1nM, 10nM, 1uM
C1-8: Melatonin 0nM, 1nM, 10nM, 1uM
 +
D1-8: Glutamate 0nM, 10nM, 100nM, 1uM
D1-8: Glutamate 0nM, 10nM, 100nM, 1uM
 +
E1-8: Orphanin FQ 0nM, 1nM, 10nM, 100nM
E1-8: Orphanin FQ 0nM, 1nM, 10nM, 100nM
 +
F1-8: Vasopressin 0nM, 10nM, 100nM, 1uM
F1-8: Vasopressin 0nM, 10nM, 100nM, 1uM
 +
G1-3: ATGR1/fMLP
G1-3: ATGR1/fMLP
 +
G4-6: AGTR2/fMLP
G4-6: AGTR2/fMLP
 +
G7-9: GRM2/fMLP
G7-9: GRM2/fMLP
 +
G10-12: CCR7/fMLP
G10-12: CCR7/fMLP
 +
H1-3: GRM4/fMLP
H1-3: GRM4/fMLP
 +
H4-6: MTNR1A/fMLP
H4-6: MTNR1A/fMLP
 +
H7-9: OPRL1/fMLP
H7-9: OPRL1/fMLP
 +
H10-12: VIB/fMLP
H10-12: VIB/fMLP
Plate #3
Plate #3
 +
A1-3: fMLP WT
A1-3: fMLP WT
 +
A4-6: Media
A4-6: Media
 +
A7-9: Input WT
A7-9: Input WT
 +
B1-3: Input AGTR1
B1-3: Input AGTR1
 +
B4-6: Input AGTR2
B4-6: Input AGTR2
 +
B7-9: Input CCR7
B7-9: Input CCR7
 +
B10-12: GRM2
B10-12: GRM2
 +
C1-3: Input GRM4
C1-3: Input GRM4
 +
C4-6: Input MTNR1A
C4-6: Input MTNR1A
 +
C7-9: Input OPRL1
C7-9: Input OPRL1
-
C10-12: Input VIB
 
 +
C10-12: Input VIB
'''07.15'''
'''07.15'''
Line 439: Line 636:
1. AGTR1: 2uL
1. AGTR1: 2uL
 +
2. AGTR2: 3uL
2. AGTR2: 3uL
 +
3. GRM2: 2uL
3. GRM2: 2uL
 +
4. GRM4: 2.3uL
4. GRM4: 2.3uL
 +
5. hM4: 1uL
5. hM4: 1uL
 +
6. MTNR1A: 2uL
6. MTNR1A: 2uL
 +
7. OPRL1: 2uL
7. OPRL1: 2uL
 +
8. pCDNA: 2uL
8. pCDNA: 2uL
 +
9. pMaxGFP: 2uL
9. pMaxGFP: 2uL
8:40
8:40
 +
5-hour incubation at 37C
5-hour incubation at 37C
Line 456: Line 662:
Chemoattractant dilutions:
Chemoattractant dilutions:
1. 10nM stock Angiotensin II
1. 10nM stock Angiotensin II
 +
[0nM, 10pM, 1nM, 10nM]
[0nM, 10pM, 1nM, 10nM]
2. 500uM stock Orphanin FQ
2. 500uM stock Orphanin FQ
 +
[0nM, 1nM, 10nM, 100nM]
[0nM, 1nM, 10nM, 100nM]
3.    50mM stock Glutamate
3.    50mM stock Glutamate
 +
[0nM, 10nM, 100nM, 1uM]
[0nM, 10nM, 100nM, 1uM]
4. 100mM stock Melatonin
4. 100mM stock Melatonin
 +
[0nM, 1nM, 10nM, 1uM]
[0nM, 1nM, 10nM, 1uM]
Line 470: Line 680:
1.    Minipreps (249.4 ng/uL)
1.    Minipreps (249.4 ng/uL)
 +
2.    Digestion, run gel
2.    Digestion, run gel
-
3.    FACs Analysis
 
 +
3.    FACs Analysis
'''07.08'''
'''07.08'''
Line 479: Line 690:
In 5-day-differntiated HL-60:
In 5-day-differntiated HL-60:
 +
1.    L1+pMaxGFP
1.    L1+pMaxGFP
 +
2.    L2+pMaxGFP
2.    L2+pMaxGFP
 +
3.    L3+pMaxGFP
3.    L3+pMaxGFP
 +
4.    M1+pMaxGFP
4.    M1+pMaxGFP
 +
5.    M2+pMaxGFP
5.    M2+pMaxGFP
 +
6.    H1+pMaxGFP
6.    H1+pMaxGFP
 +
7.    H2+pMaxGFP
7.    H2+pMaxGFP
 +
8.    H3+pMaxGFP
8.    H3+pMaxGFP
-
 
II.    Transwell Assay
II.    Transwell Assay
Chemoattractant dilution:
Chemoattractant dilution:
 +
1.    10mM stock CNO
1.    10mM stock CNO
Line 499: Line 718:
In 5-day-differentiated HL-60:
In 5-day-differentiated HL-60:
 +
1.    hM2+pMaxGFP
1.    hM2+pMaxGFP
 +
2.    hM3+pMaxGFP
2.    hM3+pMaxGFP
 +
3.    hM4+pMaxGFP
3.    hM4+pMaxGFP
 +
4.    pCDNA3.1+pMaxGFP
4.    pCDNA3.1+pMaxGFP
*Unnecessary second spin for samples
*Unnecessary second spin for samples
 +
4-hour incubation at 37C
4-hour incubation at 37C
Line 510: Line 734:
12-well plates: CNO (light sensitive)
12-well plates: CNO (light sensitive)
-
0nM 10nM 100nM 1uM
 
-
0nM 10nM 100nM 1uM
 
-
0nM 10nM 100nM 1uM
 
Control plate: 10nM fMLP
Control plate: 10nM fMLP
-
hM2 hM3 hM4 pCDNA
 
-
hM2 hM3 hM4 pCDNA
 
-
hM2 hM3 hM4 pCDNA
 
Chemoattractant dilutions:
Chemoattractant dilutions:
 +
1.    10mM stock CNO
1.    10mM stock CNO
Line 525: Line 744:
Cell dilutions: 100,000cells/400uL (8mL)
Cell dilutions: 100,000cells/400uL (8mL)
 +
1.    pCDNA
1.    pCDNA
 +
2.65mL cells + 5.35mL Media
2.65mL cells + 5.35mL Media
2.    hM2
2.    hM2
 +
4.3mL cells + 3.7mL Media
4.3mL cells + 3.7mL Media
3.    hM3
3.    hM3
 +
3.88mL cells + 4.12mL Media
3.88mL cells + 4.12mL Media
4.    hM4
4.    hM4
 +
3.85mL cells + 4.15mL Media
3.85mL cells + 4.15mL Media
30-minute incubation at 37C
30-minute incubation at 37C
 +
Remove wells; add 12uL EDTA to each sample
Remove wells; add 12uL EDTA to each sample
 +
Pipet samples into eppendorfs
Pipet samples into eppendorfs
 +
Pipet 100uL into 96-well plate
Pipet 100uL into 96-well plate
 +
     +  100uL fixation    +    25uL beads
     +  100uL fixation    +    25uL beads
96-well plate
96-well plate
 +
A: hM2 CNO
A: hM2 CNO
 +
B: hM3 CNO
B: hM3 CNO
 +
C: hM4 CNO
C: hM4 CNO
 +
D: pCDNA CNO
D: pCDNA CNO
 +
E: hM2 fMLP, hM3 fMLP, hM4 fMLP, pCDNA fMLP
E: hM2 fMLP, hM3 fMLP, hM4 fMLP, pCDNA fMLP
 +
F: hM2 input cells, hM3 input cells, hM4 input cells
F: hM2 input cells, hM3 input cells, hM4 input cells
 +
G: control input cells
G: control input cells
Line 557: Line 792:
Materials
Materials
 +
•    5 x 106 5-day-differentiated HL-60 per transfection
•    5 x 106 5-day-differentiated HL-60 per transfection
 +
•    2.5mL IMDM media from Gibco per reaction
•    2.5mL IMDM media from Gibco per reaction
 +
•    Amaxa Nucleofactor Kit V. Premix solutions and date bottle (good for 2 months)
•    Amaxa Nucleofactor Kit V. Premix solutions and date bottle (good for 2 months)
 +
•    24-well cell culture plates
•    24-well cell culture plates
 +
•    1.5mL eppendorfs
•    1.5mL eppendorfs
Protocol
Protocol
 +
1.    Check cells under light microscope to make sure they look healthy (morphological polarity).
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.
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.
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.
4.    During centrifuge spin, turn on Amaxa Nucleofactor Device and select program Y-001.
 +
5.    Prepare transfection reagents.
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.
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.
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.
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.  
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.
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.
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.
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.
13.    Let recover/express for 2-6 hours. Use FACs or microscope to check expression levels.
Line 583: Line 836:
  Alpha-Pix   
  Alpha-Pix   
 +
  B2AR   
  B2AR   
 +
  BPRX-GFP-Vasp   
  BPRX-GFP-Vasp   
 +
  DOR-Erm   
  DOR-Erm   
 +
  DOR-Ezrin   
  DOR-Ezrin   
 +
  GFP-Vasp-BPRX   
  GFP-Vasp-BPRX   
 +
  Intersectin   
  Intersectin   
 +
  LPD 775-1250   
  LPD 775-1250   
 +
  P-Rex 5   
  P-Rex 5   
IV.    GPCR Organization
IV.    GPCR Organization
-
 
V.    Transformations
V.    Transformations
  ActA 30-612   
  ActA 30-612   
-
  ActA 225-392   
+
   
 +
ActA 225-392   
 +
 
  B2AR Actinin   
  B2AR Actinin   
 +
  Beta-Pix   
  Beta-Pix   
 +
  DOR-Actinin   
  DOR-Actinin   
  DOR-Kifc   
  DOR-Kifc   
 +
  LPD 775-1250   
  LPD 775-1250   
 +
  Vav   
  Vav   
Line 615: Line 881:
P1: Live cell population
P1: Live cell population
 +
P2: Beads
P2: Beads
 +
P3: Live green cell population
P3: Live green cell population
Corrected live green cell population
Corrected live green cell population
 +
# cells acquired/# beads acquire x 11,111 beads
# cells acquired/# beads acquire x 11,111 beads
Migration (%)
Migration (%)
 +
# cells acquired/average # input cells x 100
# cells acquired/average # input cells x 100
-
07.01  
+
'''07.01'''
I.    Transwell Assay
I.    Transwell Assay
Protocol
Protocol
 +
1.    Label 12-well plates.
1.    Label 12-well plates.
 +
2.    Sigmacote 12-well plates.
2.    Sigmacote 12-well plates.
 +
3.    Filter cells.
3.    Filter cells.
 +
4.    Count cells.
4.    Count cells.
 +
5.    Centrifuge cells: 1000rpm for 5 minutes.
5.    Centrifuge cells: 1000rpm for 5 minutes.
 +
6.    Dilute chemoattractants.
6.    Dilute chemoattractants.
 +
7.    Aliquot 1mL/well.
7.    Aliquot 1mL/well.
 +
8.    Aspirate.
8.    Aspirate.
 +
9.    Resuspend with 5mL media.
9.    Resuspend with 5mL media.
 +
10.    Dilute cells: 250,000/mL. 250,000x7mL = 1,750,000 cell
10.    Dilute cells: 250,000/mL. 250,000x7mL = 1,750,000 cell
 +
11.    Place inserts into each well.
11.    Place inserts into each well.
 +
12.    Pipet 400uL cells into each insert.
12.    Pipet 400uL cells into each insert.
 +
13.    Incubate: 37C, 5% CO2 for 30 minutes.
13.    Incubate: 37C, 5% CO2 for 30 minutes.
 +
14.    Pipet 12uL 0.5M EDTA into each well.
14.    Pipet 12uL 0.5M EDTA into each well.
 +
15.    Tap plate, remove inserts.
15.    Tap plate, remove inserts.
 +
16.    Resuspend solution, transfer to eppendorf.
16.    Resuspend solution, transfer to eppendorf.
 +
17.    Transfer 100uL into  96-well plate.
17.    Transfer 100uL into  96-well plate.
 +
25uL beads + 100uL Fixation Buffer + 100uL cells
25uL beads + 100uL Fixation Buffer + 100uL cells
 +
or 25uL Media + 100uL Fixation Buffer + 100uL cells
or 25uL Media + 100uL Fixation Buffer + 100uL cells
Line 663: Line 952:
fLMP: 10nM stock solution
fLMP: 10nM stock solution
 +
1uM → 100nM → 10nM → 0nM
1uM → 100nM → 10nM → 0nM

Latest revision as of 03:47, 22 October 2009

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