7.2: Calcium

7.2: Calcium kdorfman Wed, 10/26/2011 - 19:05

Release of Internal Calcium Stores

Wednesday, 11/2/11

Rescheduled for Monday, 11/7/11, due to snowday 10/31/11

7.1: External stimuli

7.1: External stimuli kdorfman Wed, 10/26/2011 - 16:08

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

Cells for 7.1 kdorfman Wed, 10/26/2011 - 16:12

3t3 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 - 16:13

HBS

HBS kdorfman Wed, 10/26/2011 - 16:15

HBS 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 - 16:26

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

ATP kdorfman Wed, 10/26/2011 - 17:13

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

Bradykinin kdorfman Wed, 10/26/2011 - 17:22

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 kdorfman Wed, 10/26/2011 - 16:22

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

Vasopressin kdorfman Wed, 10/26/2011 - 18:29

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

test drop downs for 7.1 reagents kdorfman Thu, 10/27/2011 - 16:55

This page tests the use of collapsible sections for recipe pages

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

10 µM solution for use in HBS

Make 24 aliquots of 1.6 mL (16 for Lab 7.1, 8 for Lab 7.2

Cells for 7.2

Cells for 7.2 kdorfman Wed, 10/26/2011 - 19:07

3t3 on large-diameter MatTek dishes

4 dishes per group, plus extra = 32

Plate on Monday before Wednesday lab, or Saturday if it's on Monday

Reagents for 7.2

Reagents for 7.2 kdorfman Wed, 10/26/2011 - 19:08

Use left-over aliquots of stimulants from 7.1 with calcium, make calcium-free reagents for 7.2

ATP

ATP kdorfman Wed, 10/26/2011 - 19:23

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 (=5.731 mg/L) ATP in HBS

Use ATP aliquots from Lab 7.1

10 µM ATP, Calcium-free

Lab 7.2: 0.75 mL per coverslip x 2 coverslips x 7 groups = 10.5 mL

Make 13 mL
13 µL 10 mM ATP stock in 13 mL ca- HBS (1 µL 10 mM ATP stock per 1 mL HBS.)

8 1.6 mL aliquots for Lab 7.2

Bradykinin

Bradykinin kdorfman Wed, 10/26/2011 - 19:25

1mg/mL Bradykinin stock
(= 1.0602 mM) Sigma B3259 - 1 mg septum bottle
MW = 106.02
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)

2 µM Bradykinin in HBS

Use bradykinin aliquots from Lab 7.1

2 µM Bradykinin, Calcium-free
(=2.12 µg/mL)

Lab 7.2: 0.75 mL per coverslip x 2 coverslips x 7 groups = 10.5 mL

Frozen aliquots are 21.2 µL 1 mg/mL. Makes 10 mL

~2 µL 1 mg/mL stock per 1 mL working solution

21.2 µL 1 mg/mL stock in 10 mL Ca-free HBS

Make 6 aliquots of 1.6 mL

Make more if necessary. (Aliquots should have been enough for 15 mL?)

Fluo-4

Fluo-4 kdorfman Wed, 10/26/2011 - 19:17

Use Fluo-4 aliquots from Lab 7.1

HBS

HBS kdorfman Wed, 10/26/2011 - 19:14

HBS needed for rinsing and to make other solutions

(3 rinses + 1 sham dilution) x 4 expts/grp x 1 mL = 16 mL

Aliquot 25 mL per group

300 mL HBS

300 mL Ca-free HBS

Ionomycin

Ionomycin kdorfman Wed, 10/26/2011 - 19:41

Ionomycin stock

1 mM in DMSO or EtOH. 1 mg makes 1.34 mL stock.

3 µM Ionomycin in HBS
Dilute stock 1:333 in HBS: 3 µL/1 mL

21 µL in 7 mL, aliquot 800 µL

They only need 750µL, because they do one experiment in HBS, then two in Ca-free HBS.

3 µM Ionomycin in Ca-free HBS
Dilute stock 1:333 in Ca-free HBS: 3 µL/1 mL
39 µL in 13 mL

Aliquot 1.55 mL so they can do two 750 µL experiments

Vasopressin

Vasopressin kdorfman Wed, 10/26/2011 - 19:34

1 mg/mL (= 0.9225 mM) Vasopressin stock in water

1 µM dilution to make student solutions

1 µL 1 mg/mL in 922.5 µL water (NO CALCIUM, so stock can be used for Ca-free solutions )

10 nM (= 1.084 µg/mL) Vasopressin in HBS for students

Use Vasopressin aliquots from Lab 7.1

Calcium Free Vasopressin 10 nM

10 nM (= 1.084 µg/mL) for students

1 part 1µM dilution to 99 parts Ca-free HBS

Lab 7.2: 0.75 mL per coverslip x 2 coverslips x 7 groups = 10.5 mL

Make 13 mL: 130 µL + 13 mL Ca-free HBS

Make 8 aliquots of 1.6 mL