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