Flow-Cytometric Immune Function Methodology(14)

54 Willmann

4. Notes

The low frequency of peripheral blood DCs and indirect staining procedures are creating technical challenges in the detection of DCs in unseparated samples. As demonstrated with these protocols and associated results shown inFigs. 5–7, it is possible to perform these tasks in a step-by-step procedure. Beyond the information already provided, the author would like to share a few additional comments for a successful performance of the assay:

1. The inability to detect a cytokine signal may be the result of insufficient

permeabilization or inactive BFA or LPS solutions. Incorrect storage conditions may inactivate LPS or BFA solutions. It is imperative that correct dilutions and concentrations for solutions like FACS Permeabilizing, BFA, or LPS are used. Blood drawn in other anticoagulants than sodium heparin should not be used. The assay is incompatible with EDTA or ACD anticoagulants.

2. It might be necessary to transfer processed samples to polystyrene tubes for

the ow-cytometric acquisition. Polypropylene tubes do not always t tightly onto the sample injection system of the ow cytometer. Perform the transfer right before acquisition.

3. Dendritic cells have a slightly decreased density over other leukocytes, which

can lead to a loss of DCs by insufficient pelleting during cell processing steps. Additionally, cell permeabilization procedures decrease the density of leukocytes. Therefore, if the yield of DCs appears too low, consider prolonging the centrifugation time or increasing the g-force of the centrifugation in

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