Leukocytes are the main cells of immune system, but also contribute to other systems and participate in pathogenesis of different diseases. In particular, leukocytes are involved in the progression of diabetic retinopathy due to their hyperactivation in diabetes. However, a connection between diabetes and the dysfunction of leukocytes is poorly understood. For a more complete picture, studies of the leukocytes activation under the influence of various substances are necessary. Arachidonic acid (AA) and its metabolites are the strongest activating factors of leukocytes. However, the studies involving AA are complicated because it is water-insoluble. Here we describe the method to study activation using photolabile analogs of AA.
Platelets are the most important participants in both normal hemostasis and pathological thrombotic process. Platelet activation needs to be studied today because management of this process is the key to progress in the treatment of atherosclerotic cardiovascular diseases. Evaluating platelet activation at the single-cell level is a promising approach for investigating platelet functions, as well as studying the action of various receptors. Previously such single-cell studies were conducted by the immobilization of platelets on the surface, which changes the platelet signaling significantly . In this paper, we describe several activation methods to overcome this limitation, in particular, by use of photolabile “caged” analogues of activation agonists. Activation can be initiated by optical pulse with the duration of tens of milliseconds. Therefore, the technique allows one to track the very early stage of activation in freely moving single platelets. In particular, it enables the assessment of the delay between the stimulus and the calcium response in platelets. The proposed method can be used for in-depth studies of platelet physiology.