7.1: External stimuli

Cellular Responses to External Stimuli
Monday, 10/31/11

Rescheduled for 11/2/11 because of storm on 10/31/11

Cells for 7.1

3t3 on large diameter MatTek dishes

4 dishes per group plus extra = 32

Plate 2 days before class.

Reagents for 7.1

HBS

HBS for washes and making solutions

25 mL per group

(total = 175 mL)

Make from 10X

Make 350 mL

Pluronic F-127

Stock

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

10 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

1 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 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

1 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

This page tests the use of collapsible sections for recipe pages