Re: DNA stain for malariaGabriella,
We have a 488 nM argon laser on FACScan. We detect DRAQ stained RBCs on FL3 and other RBC markers of interest on FL1. We usually do not keep cells passed 72 h after staining and fixing (1 % paraformaldehyde). I would imagine that one can keep fixed DRAQ stained RBCs at 4 degrees for weeks. We always wash the cells after staining.
John
----- Original Message -----
From: Gabriel Alespeiti
To: Dr. John Waitumbi
Cc: cytometry@flowcyt.cyto.purdue.edu
Sent: Tuesday, July 30, 2002 7:39 PM
Subject: Re: DNA stain for malaria
John,
Thank you very much for your reply. This is the type of response I was looking for. Biostatus says that DRAQ5 has an excitation maximum at 647nm and despite sub-optimal excitation at 488nm wavelength, the high affinity binding characteristics of DRQ5 permit DNA content reporting by 488 nm laser flow cytometry. Which wavelength are you using for parasite detection?
It seems you recommend me to stain and then fix with formaldehyde. Since DRAQ5 is stable at room temperature and is not duly affected by light; based on your advice, I would stain the sample directly with DRAQ5 (10uM final concentration / 5 minutes) and then fix with formaldehyde (1-4%w/v).
Stored at 4fC in the dark until flow analysis (no wash).
Is this protocol acceptable, or should the staining be just before analysis? Another question, do you know for how long are the samples stable with DRAQ5?
Gabriel,
We routinely use DRAQ5, a DNA stain that is sold by Biostatus (www.biostatus.com). It's rapidly taken in by living cells (5 min at room temperature) and binds to DNA stoichiometrically. We use it at concentration of 10 mM final. You should be able to stain the RBCs, fix them at source with formaldehyde and then ship them. We get good correlation between microscopy parasitemia and flow. The staining is however not reliable for parasitemias less than 1%.
John Waitumbi
----- Original Message -----
From: Gabriel Alespeiti
To: Cytometry Mailing List
Sent: Thursday, July 25, 2002 10:12 PM
Subject: Re: anti-malarial antibodies
Dear colleagues,
I have read the replies on anti-malarial antibodies and I hope to get some help from the experts in this field. A researcher in the Institute is conducting parasitaemia studies of Plasmodium falciparum infected erythrocytes using Giemsa stained microscopy, a tedious, time consuming and technically imprecise method. We want to replace the microscopic counts with a flow cytometry method. Peripheral blood will be collected in Brazil and will need to be fixed before shipping. A FACScalibur is available for this work. We would like to analyze 25 samples a day, the expected parasitemia level is between 0.05% and 8%.
I tried formaldehyde fixation followed by propidium iodide staining and even though there is a direct correlation between cytometric and microscopic determination, the manual counts are significantly higher than the flow counts.
What I'm looking for is a simple, proven protocol for daily use that gives directly the % of parasitaemia by flow. In our case, determination of erythrocyte or parasite antigens; even though useful, is not necessary. What is important for us is to be able to detect low infection levels.
Does anybody know, what is the lowest level of infection that can be detected by flow?
Please post the reply on this site, so other people can benefit from it. Thank you very much.
--
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Gabriel E. Alespeiti
Supervisor, Flow Cytometry
Lindsley F. Kimball Research Institute
New York Blood Center
310 E.67th St. Room 2-16E
New York, NY 10021
Phone: (212) 570-3346
Fax: (212) 570-3307
E-mail:gabriel_alespeiti@nybc.org
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