A disposable-chamber temperature-regulation system for the study of intracellular calcium levels in single live T cells using fluorescence digital-imaging microscopy.
Utilizing flow cytometry, we previously demonstrated that the potassium channel blocker margatoxin (MgTX) inhibits the [Ca2+]i transient involved in T-cell activation. We wished to extend these studies to single-cell transients using florescence digital-imaging microscopy (DIM). However, the most currently available temperature-regulation chambers reuse part or all of the apparatus and introduce compounds via perfusion. Thus, these apparatuses are not suitable for studies involving compounds that are particularly sticky. We have designed a dual-temperature regulation system that will maintain Nunc, eight-well, coverglass-bottom, disposable chambers, and three disposable addition pipets at 37 degrees C for physiological studies on an inverted digital-imaging microscope. We have demonstrated that calcium transients of human T lymphocytes can be initiated and monitored reproducibly during the addition of three distinct chemical species. The DIM results correlate with flow cytometry measurements in the number of responding cells and the heterogeneity of the response in both control and MgTX-inhibited cultures. Additionally, DIM revealed that the [Ca2+]i transient is more rapid than the flow-cytometric measurement indicated. The correlation between flow cytometry and DIM permits the amalgamation of these results in the interpretation of studies on the regulation of T-cell activation.
Department of Immunology Research (R.C.B., A.S., T.B., Merck Research Laboratories, Rahway, New Jersey 07065.
R C
RC
Boltz
A
A
Sirotina
T
T
Blake
G
G
Kath
B
B
Uhrig
J
J
McKeel
C
C
Quinn
D002118:Calcium / Q000378:metabolism*; D005434:Flow Cytometry; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000295:instrumentation*; D066298:In Vitro Techniques; D007424:Intracellular Fluid / Q000187:drug effects / Q000378:metabolism; D007700:Kinetics; D008213:Lymphocyte Activation; D008856:Microscopy, Fluorescence / Q000295:instrumentation*; D009498:Neurotoxins / Q000494:pharmacology; D026902:Potassium Channel Blockers; D012604:Scorpion Venoms; D013601:T-Lymphocytes / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D013696:Temperature
D009498:Neurotoxins; D026902:Potassium Channel Blockers; D012604:Scorpion Venoms; C077910:margatoxin; D002118:Calcium