8: Projects

8: Projects kdorfman Tue, 11/22/2011 - 14:20

Projects

Have students make a class poster for the bulletin board in the hall.

ER-tracker

ER-tracker kdorfman Tue, 11/22/2011 - 14:22

ER-Tracker from Invitrogen

E34250 ER-Tracker™ Red (BODIPY® TR glibenclamide) for live-cell imaging 100 μg

1mM Stock

Add 110 µL DMSO to the 100 µg in vial.

  • Aliquot 1 µL

  • Freeze

1 µM working concentration

  • add 0.999 mL HBSS/Ca/Mg

Protocol

  • rinse with warm HBSS

  • treat with 1 mL warm 1 µM ER-tracker

  • incubate 15-30 min at 37C

To view stained cells

  • rinse with medium

  • view with fluorescence microscope

To fix cells

  • 4% formaldehyde, for 2 min, at 37C

  • rinse 2x in buffer - NO TRITON X-100

er-tracker.pdf

HBSS/Ca/Mg

HBSS/Ca/Mg kdorfman Wed, 11/23/2011 - 20:08

Hank's Balanced Salt Solution

Gibco cat. #14025-092

To make 1 Liter from dry ingredients

Components MW (mg/L) mM
Calcium Chloride (CaCl2) (dihyd.) 147 185 1.26
Magnesium Chloride (MgCl2-6H2O) 203 100 0.493
Magnesium Sulfate (MgSO4-7H2O) 246 100 0.407
Potassium Chloride (KCl) 75 400 5.33
Potassium Phosphate monobasic (KH2PO4) 136 60 0.441
Sodium Bicarbonate (NaHCO3) 84 350 4.17
Sodium Chloride (NaCl) 58 8000 137.93
Sodium Phosphate dibasic (Na2HPO4-6H20) 268 90.6 0.338
D-Glucose (Dextrose) 180 1000 5.56

Filter sterilize


To make from stock solutions:

Components Ci (M) cf (mM) 1000 500 250 mL
CaCl2 0.5 1.26 2.52 1.26 0.63 mL
MgCl2 1 0.493 0.493 0.247 0.108 mL
MgSO4 1 0.407 0.407 0.204 0.102 mL
KCl 1 5.33 5.33 2.67 0.131 mL
KH2PO4 1 0.441 0.441 0.22 0.11 mL
NaHCO3 0.5 4.17 8.34 4.17 0.209 mL
NaCl 5 137.93 27.59 13.79 6.9 mL
Na2HPO4 1 0.338 0.338 0.169 0.085 mL
Glucose - - 1 0.5 0.25 g

Filter sterilize

Latrunculin B

Latrunculin B kdorfman Wed, 11/30/2011 - 18:21

Alexis Biochemicals 350-036-C100 Catalog # t110

Inhibits actin polymerization and disrupts microfilament organization, as well as microfilament-mediated processes

Reported to be 10 to 100-fold more potent than the cytochalasins. May act more slowly, though. Lots of variation between cell lines.

Inactivated by FBS. (Rinse medium off before treatment.)

MW = 395.5

Soluble in DMSO and ethanol.

Store in freezer.

Active concentration range: 90 nm ( =~0.1µM) to 2.5 µM

(2.5 µM = 25 x 100 nm)

Stock is 1mm

µL 1mm stock µL serum-free buffer or medium µM final concentration
1 * 99 10
1 399 2.5
1 999 1

*Use this to make further dilutions

t110.pdf