By Myriam Consuelo Lopez
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Extra resources for ATLAS DE PARASITOLOGIA
They can be stained on live and on fixed cells, depending on the fixation protocol, and can be used to isolate live cells by affinity-label-based cell sorting with MACS or FACS. lntracellular molecules Many molecules are only present within the cell and not on its surface. As currently available affinity labels, such as antibodies, cannot penetrate intact cell membranes, intracellular molecules are not accessible in live cells. Intracellular molecules of interest include molecules involved in signal transduction, gene regulation, biochemical processes and also molecules en route to secretion or surface display.
This has to be taken into account, especially when making quantitative comparisons of the labelling intensities of different samples. 33 Concentration of reagents The concentration of the staining reagents has the strongest influence on the signal intensity versus the background labelling. Reagents should be used at optimal concentrations with the highest possible signal to noise ratio. Too low concentrations result in incomplete staining and low signal intensity, and thus yield suboptimal discrimination of positive and negative cells.
Such cells are specifically stained for the same cytokine intracellularly, and only these cells secrete this cytokine, as has been shown by sorting, subsequent culture and ELISA. This demonstrates the comparable sensitivities of these techniques. Standard protocol for intracellular staining of murine cytokines Antibodies for intracellular staining of murine cytokines See Table 2. Fixation 1. Wash cells once with PBS and resuspend in PBS at 2x 1O6rnl-'. 2. Add 1 volume of 4% formaldehyde/PBS. Incubate for 20 min at room temperature.