7.1: External stimuli
7.1: External stimuli kdorfman Wed, 10/26/2011 - 16:08Cellular Responses to External Stimuli
Monday, 10/31/11
Rescheduled for 11/2/11 because of storm on 10/31/11
Cells for 7.1
Cells for 7.1 kdorfman Wed, 10/26/2011 - 16:123t3 on large diameter MatTek dishes
4 dishes per group plus extra = 32
Plate 2 days before class.
Reagents for 7.1
Reagents for 7.1 kdorfman Wed, 10/26/2011 - 16:13HBS
HBS kdorfman Wed, 10/26/2011 - 16:15HBS for washes and making solutions
25 mL per group
(total = 175 mL)
Make from 10X
Make 350 mL
Pluronic F-127
Pluronic F-127 kdorfman Wed, 10/26/2011 - 16:26Stock
comes as dry granules - store on shelf
Also comes as solution Fisher 50-310-493
PROMOCELL GMBH PLURONICF-127 20%IN DMSO 1ML $44
Make 0.5 mL with 0.1g (= 200 mg/mL =20% w/v) in DMSO
10 µL aliquots - each makes 10 mL 0.02%
Store in freezer.
ATP
ATP kdorfman Wed, 10/26/2011 - 17:1310 mM ATP stock (from Maniotis)
60 mg ATP in 8 mL H2O
pH to 7.0 with 0.1 M NaOH
Bring volume to 10 mL with H2O
Aliquot and freeze.
10 µM solution for use (=5.731 mg/L) in HBS
Make enough for Labs 7.1 and 7.2:
Lab 7.1: 0.750 mL per coverslip x 4 coverslips x 7 groups = 21 mL
Lab 7.2: 0.75 mL per coverslip x 2 coverslips x 7 groups = 10.5 mL
Make 40 mL
40 µL 10 mM ATP stock in 40 mL HBS
(1 µL 10 mM ATP stock per 1 mL HBS.)
Make 24 aliquots of 1.6 mL (16 for Lab 7.1, 8 for Lab 7.2
Bradykinin
Bradykinin kdorfman Wed, 10/26/2011 - 17:221 mg/mL stock
(= 1.0602 mM)
Sigma B3259 - 1 mg septum bottle
MW = 1060.2
Add 1 mL H2O to the 1 mg in the bottle (NO CALCIUM! - some will be used for the calcium experiments in Lab 7.2)
Aliquots are 21.2 µL.
2 µM solution for use
(=2.12 µg/mL)
NOTE: Rather weak response in 2011. Try doubling the concentration?
Make enough for Labs 7.1 and 7.2:
- Lab 7.1: 0.750 mL per coverslip x 4 coverslips x 7 groups = 21 mL
- Lab 7.2: 0.75 mL per coverslip x 2 coverslips x 7 groups = 10.5 mL
Make 40 mL
~2 µL 1 mg/mL stock per 1 mL working solution
Each 21.2 µL aliquot of 1 mg/mL makes 10 mL 2µM working solution
84.8 µL 1 mg/mL stock in 40 mL HBS
Make 24 aliquots of 1.6 mL (16 for Lab 7.1, 8 for Lab 7.2)
4 µM solution for use
(=4.24 µg/mL)
NOTE: Double the 2011 concentration
Make enough for Labs 7.1 and 7.2:
- Lab 7.1: 0.750 mL per coverslip x 4 coverslips x 7 groups = 21 mL
- Lab 7.2: 0.75 mL per coverslip x 2 coverslips x 7 groups = 10.5 mL
Make 40 mL
~4 µL 1 mg/mL stock per 1 mL working solution
Each 21.2 µL aliquot of 1 mg/mL makes 5 mL 2µM working solution
169.6 µL 1 mg/mL stock in 40 mL HBS (8 aliquots)
Make 24 aliquots of 1.6 mL (16 for Lab 7.1, 8 for Lab 7.2)
Fluo-4
Fluo-4 kdorfman Wed, 10/26/2011 - 16:22Fluo-4 stock
Invitrogen F14217 500 µL
1mM in DMSO
Protect from light
Store in dissicator.
20 µL aliquots. Each makes 10 mL of 2 µM solution
Fluo-4 staining solution
2 µM Fluo-4 + 0.02% pluronic in HBS
Make enough for 7.1 and 7.2: 250 µL x 4 expts x 8 grps x 2 days = 16 mL.
Make 20 mL
Need 16 aliquots, each ~1.05 mL
| ingredient | 5 | 10 | 15 | 20 | mL |
|---|---|---|---|---|---|
| 1 mM Fluo-4 stock | 10 | 20 | 30 | 40 | µL |
| 20 % w/v Pluoronic | 5 | 10 | 15 | 20 | µL |
plus HBS to final volume
Vasopressin
Vasopressin kdorfman Wed, 10/26/2011 - 18:291 mM stock in water
(NO CALCIUM, so stock can be used for Ca-free solutions in Lab 7.2)
1 mg in bottle, MW = 1084
Add 0.92 mL to make 1mM stock
1 µL aliquots in freezer
1 µM dilution to make student solutions
1 µL 1 mM in 999 µL water
(NO CALCIUM, so stock can be used for Ca-free solutions in Lab 7.2)
10 nM (= 1.084 µg/mL) for students
NOTE: Rather weak response in 2011. Try doubling the concentration?
1 part 1µM dilution to 99 parts HBS
Make enough for Labs 7.1 and 7.2: Lab 7.1: 0.750 mL per coverslip x 4 coverslips x 7 groups = 21 mL Lab 7.2: 0.75 mL per coverslip x 2 coverslips x 7 groups = 10.5 mL
Make 40 mL: 400 µL 1µM Vasopressin + 39.6 mL HBS
Make 24 aliquots of 1.6 mL (16 for Lab 7.1, 8 for Lab 7.2
test drop downs for 7.1 reagents
test drop downs for 7.1 reagents kdorfman Thu, 10/27/2011 - 16:55This page tests the use of collapsible sections for recipe pages