BrdU staining and surface antigen

Gael Dulude nomeansno2 at yahoo.ca
Mon Jun 7 21:50:03 EST 2004


Hi,

I have tried the BD kit and it works fine but it is very expensive. You  
can try two other protocols wich will give you the same results for a lot  
less money:

The antibody I used was from pharmingen:
Set Catalog Number: 556028 (Was: 36634K)
Description: FITC-conjugated antibody set

First protocol was modified from (Bruno Lucas, Florence Vasseur, and  
Claude Penit
journal of Immunology, 1993, 151 : 4574.)

Double stained cells were fixed in 200 ul of 1% paraformaldehyde  
containing 0.01% Tween 20 (Sigma) for 24 to 48 h at 4°C in the dark,  
Overnight : 12-16h

Cells were washed in PBS and then in 4.2 mM MgCl2/0.15 M NaCl, pH 5. and  
thereafter incubated for 1 h at room temps in the same buffer containing  
50 Kunitz units of bovine pancreatic deoxyribonuclease I (1 ml).

After a new wash in PBS, thymocytes were incubated in PBS containing 0.5%  
Tween 20 and anti-BrdUrd mAb, 30 min at room temperature in the dark.  
(2x106cell. = 15ul Ab: 100ul PBS) amd then wash with PBS.

Second protocol can be found in current protocol in immunology and it  
comes from (Tough, D.F., and J. Sprent. 1994. Turnover of naive- and  
memory-phenotype T cells. J. Exp. Med. 179:1127?1135.)It use alcool  
fixation instead of HCL.

Measurement of T and B Cell Turnover with Bromodeoxyuridine
BASIC PROTOCOL:  MEASUREMENT OF T AND B CELL TURNOVER WITH  
BROMODEOXYURIDINE

Materials
Experimental animals
0.8 mg/ml BrdU (Sigma) in water (for oral administration) or 4 mg/ml BrdU  
in PBS (for injection)

0.15 M NaCl, ice cold
95% ethanol, ice cold
Anti-BrdU-FITC (Becton Dickinson Immunocytometry)

Paraformaldehyde solution
Dissolve 1% (w/v) paraformaldehyde and 0.01% (v/v) Tween 20 in PBS. Stir  
at low heat (70C) to dissolve paraformaldehyde. Store up to 6 months at  
room temperature. Filter through a 0.2-um before use.

DNase I solution
Dissolve deoxyribonuclease I (from bovine pancreas; Sigma) to 50 Kunitz  
U/ml in 4.2 mM MgCl2/0.15 M NaCl, pH 5. Make solution fresh before use or  
make in concentrated form (e.g., 500 U/ml) and store up to 2 months at	
-20C.

Treat animals with BrdU

1. Administer BrdU to animals in drinking water (dissolve in sterile  
drinking water at 0.8 mg/ml) or by i.v. or i.p. injection (dissolve in PBS  
to 4 mg/ml, then inject 0.2 ml--0.8 mg--per mouse as described in UNIT	
1.6).

Because BrdU is light sensitive, it is necessary to prepare fresh  
BrdU-containing drinking water daily. Injection may be used in conjunction  
with administration in drinkingwater when the exact start time of BrdU	
treatment is critical. Injecting BrdU either i.m. or i.p. is preferable  
when a short pulse of BrdU labeling is desired.

Prepare cells and stain for surface markers
2. Collect lymphoid organs and make single-cell suspensions. Prepare cells  
for immunofluorescence.
3. Stain cells for surface markers using appropriate antibodies and  
fluorochromes .
4. Wash cells by adding 1 ml PBS, then centrifuging 6 min at 300g, 4C.
5. Pour off all supernatant and resuspend cells in 0.5 ml ice-cold 0.15 M  
NaCl.

Fix cells
6. While gently vortexing cells, add 1.2 ml ice-cold 95% ethanol dropwise.  
Incubate cells 30 min on ice. Gentle vortexing of the cells and slow  
addition of ethanol help prevent cell clumping.
7. Wash cells by adding 2 ml PBS, then centrifuging 6 min at 450g, 4C.

Because ethanol-fixed cells do not form a tight pellet, it is necessary to  
centrifuge at a higher speed during all washing steps from this point  
onward to minimize cell loss.

8. Pour off supernatant. Resuspend cells in 1 ml paraformaldehyde  
fixative. Incubate 30 min at room temperature.
9. Centrifuge cells 6 min at 450g, 4C.

Stain to detect BrdU and analyze results

10. Pour off supernatant. Resuspend cells in 1 ml DNase I solution.  
Incubate 10 min at room temperature.
11. Repeat step 7.
12. Pour off supernatant. Resuspend cells and add 10 ul anti-BrdU-FITC.  
Incubate 30 min at room temperature.
13. Repeat step 7.
14. Resuspend cells in 500 ul PBS. Analyze by flow cytometry.
Samples may be analyzed immediately or stored up to 1 week protected from  
light at 4?C before being analyzed.


Good luck

On Mon, 7 Jun 2004 13:47:47 +0200, Sandra Aresta <saresta at hybrigenics.fr>  
wrote:

> Hello everybody!
>
> I'm trying to measure cell proliferation by flow cytometry in cells
> transfected with a membrane protein. However, I cannot denature my DNA
> to expose BrdU with HCl, because I loose the membrane staining. I've
> tried to use DNase instead but it has been difficult to optimise (the
> DNA gets too degraded, even with low amounts of DNase). Could anyone
> suggest a solution?
>
> I've seen that Phoenix Flow Systems sells a kit that should enable to
> combine BrdU and other antigens detection, named "Absolute-S". There is
> another kit from BD, "BrdU Flow kit", that is supposed to work as well.
> Does anyone know these, and if they work OK?
>
> Thank you in advance for your help.
>
> Sandra
>
>
>
>
>
>
> Sandra Aresta, PhD
>
> Biotechnology
>
> Hybrigenics SA
>
> 3-5 impasse Reille
>
> 75014 Paris
>
> France
>
> Tel: +33 (0) 1 58 10 38 00 / 60
>
> Fax: +33 (0) 1 58 10 38 49
>
>



-- 
Gaël Dulude, Ph.D
Laboratoire d'immunologie
Centre de Recherche du CHUM
Pavillon André Viallet, Hôpital St-Luc
Laboratoire 964
264, rue René Lévesque Est
Montréal, QC, Canada, H2X 1P1
Tel: (514) 890-8000 #35234
Email: gaeldulude at yahoo.ca



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