Immunology & Immunological Disorders Poster Session

Re: Poster 456

Diana Fagan

On Sat Dec 5, Grover wrote
>Dr. Fagan: Hope you are enjoying the meeting.  Great poster.Hope you are enjoying the meeting.  Great poster. I am intrigued by the composition of your PBS-azide. followed by a wash with PBS-azide: 0.01 M sodium phosphate and 0.015 M sodium chloride, containing 0.01g/ml of sodium azide, pH 7.4, at 4o C).  First this is hypotonic and would it not swell the cells.  Second, I understood that azide would inhibit any mitochondrial activity of the cell.  Anyways, I was intrigued by the effect of K-oxalate.  What sort of concentration of K-oxalate did you use?  I would expect high concentrations of this agent to produce cell depolarization and hence cell activation.

Dr. Grover,

Thank you for your interest in my poster. You are obviously more observant than I am, as you noticed the concentration of NaCl was incorrect. The NaCl concentration in PBS is 0.15 M, which makes the solution isotonic.

In response to your second question: The sodium azide is added to inhibit ATP generation. This is a common procedure in flow cytometry. It is intended to prevent the endocytosis of the fluorescent antibody by the cell.

In response to your third question: The potassium oxalate was 2 mg/ml. the tubes also contained 2.5 mg/ml sodium fluoride. It is very likely that cell activation did occur, resulting in the shedding of CD62 (L-selectin). It is also possible that the anticoagulants directly inhibited binding of the anti-CD62 antibody. The anticoagulants were chosen to represent those used in the Saint Elizabeth Hospital Emergency Room. We are interested in using pre-collected blood for clinical studies and were testing the effect of the various anticoagulants on cell adhesion molecule detection.

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