Hi All,
Is it possible to sort bacteria using flow? Want to start some bacterial
work but never done it before. I would like to be able to isolate anywhere
between 1-10 bacterial cells. If anyone knows of any protocol for this I
would greatly appreciate it. Thanks a lot.
sincerely,
Ragoo
^^^^
/o o\
| U |
**************---oOo------------oOo--***********************
Arumugham Raghunathan, Ph.D
Group Leader
Molecular Staging Inc.,
Tel# 203-772-5074
http://www.molecularstaging.com
http://home.talkcity.com/HotRodRd/imragoo/
*************************************************************
-----Original Message-----
From: Salinas, Richard [mailto:rsalinas@saci.org]
Sent: Tuesday, July 23, 2002 5:21 PM
To: cyto-inbox
Subject: Can't get BrdU labeling
Greetings,
I am having some trouble with my attempts at labeling cells (HL60) with
BrdU. I have not been able to see any labeling with BrdU-FITC antibody. I am
using the following basic protocol:
1. Incubate cells for 30 min/37*C with 20µL/mL of 10mM BrdU Stock. (Flasks
are foil wrapped to avoid light exposure.)
2. Spin cells, remove media supernatant.
3. Wash 2x with cold PBS.
4. Resuspend pellet and fix with cold 70% ETOH and store at 4*C in the
dark. (Usually left overnight.)
5. Centrifuge cells, remove ETOH supernatant.
6. Resuspend pellet and add 3mL 4N HCl and incubate 30 min/RT.
7. Centrifuge cells, remove HCl supernatant.
8. Resuspend pellet and wash with PBS.
9. Resuspend pellet and wash with 0.5%Tween 20 solution+PBS .
10. Centrifuge cells, remove Tween 20 supernatant.
11. Add 10µL anti-BrdU-FITC and incubate for 30min/RT/dark.
12. Wash 2x with cold PBS.
13. Resuspend pellet and add 1mL of PI in PBS.
I have seen other protocols and they are more/less the same. Can anyone
share some insight on my problem. Is there something that I am missing?
Thanks.
Richard
****************************
Richard A. Salinas
Flow Cytometry Laboratory
Dept. of Radiation Oncology
UTHSCSA
Texas Research Park
(210) 677-3882
rsalinas@saci.org
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