Immunology & Immunological Disorders Poster Session

Re^2: Poster 456


A concentration of 2 mg/ml of K-oxalate will give 20-30 mM K+ on the outside which is going to depolarize most cells and hence it could activate them.  This is what I was concerned about.
On Mon Dec 7, Diana Fagan wrote
>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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