Standard Operating Procedure

 

Intracellular BrdU Staining

 

Hazards:

BrdU (5-bromo-2-deoxyuridine) is toxic.  It is harmful by inhalation, in contact with skin, and if swallowed.  It is a mutagen and may cause heritable genetic damage.  May cause harm to the unborn child.

 

Protection:

WEAR SUITABLE PROTECTIVE CLOTHING, GLOVES AND EYE PROTECTION.

 

Waste:

Cages of animals injected with BrdU will be clearly labeled ÒBrdU - HazardousÓ. Bedding should be put in yellow bags for collection and managed as hazardous chemical waste.  Excess drinking water may be sewered by permission of the Chemical Waste Program (approved 12/20/05).  Carcasses will be double bagged and disposed of as usual (incinerated).  

 

HereÕs the waste information for BrdU:

DDC:  05S0

CAS: 59143A

Compound Name: 5=Bromo-2-deoxyurdine

EPA: NONE

RECY?: NO

Amt per container:   estimate 1mg per mouse injected or fed

 

Spill Clean-Up:

Isolate the area, dispose of all trash in hazardous waste bags and autoclave.

 

Procedure:

Notes: Mice will be given either a single 100 µl (1 mg) dose of bromodeoxyuridine i.p. or be fed it in the drinking water (0.8 mg/ml + 2% sucrose) in order to label dividing cells in vivo. 

Because BrdU is light sensitive, it is necessary to prepare fresh BrdU-containing drinking water daily.

BD Perm Wash buffer is a 10X stock.  Use at 1X. 

BrdU-Alexa 647 is from Phoenix Labs, use at 1:200.

 

1.      Stain cells with surface antibodies, as usual.

2.      Wash cells in FACs buffer.

3.      Resuspend cells with 100 µl BD Cytofix/Cytoperm buffer, 15 min, on ice.

4.      Wash cells in BD Perm wash buffer.

5.      Resuspend in 100 ul BD Cytoperm Plus buffer (or alternatively 90% FCS, 10% DMSO), 10 min, ice.

6.      Wash cells in BD Perm wash buffer.

7.      Resuspend in 100 µl BD Cytofix/Cytoperm buffer, 5 min, ice.

8.      Wash cells in BD Perm wash buffer.

9.      Dilute DNase.  Add 300 µl DNase to 750 µl 1X PBS.

10.    Resuspend cells with 70 µl diluted DNase, 1 hr, 37¼C.

11.    Wash cells in BD Perm wash buffer.

12.    Resuspend cells with 50 µl BD Perm wash buffer containing anti-BrdU Alexa 647 antibody or anti-BrdU FITC, 20 minutes, RT

13.    Resuspend in 200 µl FACs buffer.

14.    Flow.

 

 DNase I

Sigma D4513

1 mg/ml in 1X PBS

 

BrdU

Sigma B5002-100MG

10 mg/ml in 1X PBS