Fluorokine® Receptor Detection Kits are designed as alternative reagents for the detection of cell surface cytokine receptors by flow cytometry. Although the staining procedure of cells with this line of reagents is straight forward, some situations can present difficulties in data interpretation. In the table below, we attempt to point out some common problems that users of Fluorokine Receptor Detection Kits may encounter. The variable nature of cytometry instrumentation and instrument set-up can dramatically influence the quality of data generated with these reagents.
Cell surface receptor analysis involves the monitoring of protein structures that are in a dynamic equilibrium with their environment so changes in the medium such as pH, temperature, ionic strength, or presence of other proteins and/or cytokines will ultimately influence the binding properties of each labeled ligand. Lastly, the biological activity of labeled ligands can modulate a cell’s receptor density. We hope the tips below will help investigators overcome simple obstacles, but if additional help is required, we encourage you to contact our technical service department.
Problem: Cells do not stain with Fluorokine
Possible Source Test or Action Cell processing error Check all steps of the staining protocol to ensure proper cell processing Cells were left on ice too long and receptors may have been downregulated Stain fresh cells Cells may have lost or decreased receptor numbers during culture Use another receptor positive cell line to test reagent or retest same cells at different cell cycle stages
Problem: Fluorokine stains known receptor negative cells
Possible Source Test or Action Voltage being applied to photomultiplier tubes on cytometer are set too high Reduce PMT voltage until >95% of events fall in the first decade of the log scale Concentration of Fluorokine in the reaction is too high Dilute the Fluorokine and compare signal intensity against cells that do express receptors Transfected cells have upregulated other cell surface structures Test Fluorokine on non-transfected cells or on other transfectants using a different parent line
Problem: Blocking antibody does not give 100% inhibition of Fluorokine signal
Possible Source Test or Action Cells were not pre-treated with Ig to block Fc receptors Repeat the test by first treating cells with mouse or human Ig to block Fc receptors
Problem: Inhibition of Fluorokine staining with unconjugated cytokine is not 100%
Possible Source Test or Action Fluorokine present at too high concentration Repeat reaction using less Fluorokine Addition of unconjugated cytokine upregulated receptors Perform all reactions at 2 - 8° C and in the presence of metabolic blockers, e.g.: azide, cytochalasin B, etc. Numerically the inhibition is not 100% Comparison of mean fluorescence intensity between the two conditions may reveal substantial inhibition