Table 2
From the following article
Live-cell assay to detect antigen-specific CD4+ T-cell responses by CD154 expression
Pratip K Chattopadhyay, Joanne Yu & Mario Roederer
Nature Protocols 1, 1 - 6 (2006)
doi:10.1038/nprot.2006.1
Table 2. Troubleshooting table.
| Problem | Possible reason | Solution |
|---|---|---|
| No CD154 signal in any well. | Wrong number of cells plated. | Ensure cell concentration is 1–2  106 cells per well. |
| Anti-CD154 not added at optimal concentration. | Titrate anti-CD154 under the assay conditions described here. | |
| Poor anti-CD154 reagent. | Try a different vendor, clone or fluorochrome. | |
| Brefeldin A substituted for monensin (Golgi-stop). | Use monensin. The assay is not compatible with brefeldin A. | |
| Wrong concentration of monensin (Golgi-stop) used. | Ensure that final monensin concentration is 0.13  l per well. | |
| No CD154 signal in SEB-stimulated well. | Poor SEB reagent or inappropriate concentration. | Obtain new SEB or test existing material for activity. |
| No CD154 signal in peptide-costimulation well. | Suboptimal peptide concentration. | Test or titrate peptide for activity. |
| Poor costimulation. | Ensure that proper concentrations of costimulatory antibodies are added. Try alternate methods of costimulation. | |
| CD154 signal in costimulation-only well. | Well is contaminated with peptide or SEB. | Repeat experiment, eliminating all sources of contamination. |
| Nonspecific CD154 signal, not associated with high levels of dead cells. | Assay cells after longer period of stimulation (when background has stabilized). | |
| Nonspecific CD154 signal, along with high numbers of dead cells. | Ensure that correct amount of monensin is used, that concentration of DMSO in wells is not too high and that dead cells are gated out of analysis with viability marker. |
