Table 11.1.2.

Troubleshooting^a

Problem	Possible cause	Solution
Poor [3H] or [32P] labeling	Low cell viability	Determine whether cell viability is compromised via Trypan blue stain and microscopy, or via AnnexinV/7-AAD stain and FACS analysis (Barouch-Bentov et al., 2009). Add cytokines (IL-7) or other growth factors, or add low unlabeled myo-inositol amounts or more FCS (which contains myo-inositol, ~55 µM final for 10% FCS), possibly after an initial short 2- to 4-hr labeling period without exogenous inositol. Avoid extended cell incubation in microvials, where cells can become hypoxic. Increase medium and air exposure of the cells by promoting monolayer formation on the plate bottom by coating it with fibronectin or genetically engineered fibronectin-like engineered protein polymer-plus to further improve cell adhesion (Esty, 1991). Decrease labeling time. If using thymocytes, use inositol-free M199 medium to improve overall thymocyte viability. Lymphocytes can also be stimulated in PBS, HBSS/HEPES, or other solutions with physiological salt concentrations, or in antibiotic-free tissue culture media (APPENDIX 2A). Optimize red blood cell lysis by varying incubation times or lysis reagent to minimize effects on the lymphocytes. Optimize cell densities between 2 × 107 and 2 × 108 cells/ml.
Poor [3H] or [32P] labeling	Low myo-[3H] inositol uptake	Increase radiolabel amount or labeling time. Optimize cell density during labeling. Make sure that neomycin or other inositol lipid binding agents are absent from the medium. Add up to 20 mM LiCl, which inhibits phosphoinositide and IP phosphatases.
Poor [3H] or [32P] labeling	Inefficient extraction from the labeled cells	Use alternative lysis/extraction reagents such as chloroform/methanol/concentrated HCl or 10% TCA (Berridge et al., 1983; Irvine et al., 1986; Berrie et al., 2007). Maximal IP yields were obtained when lysing the cells in 10% TCA, followed by a 20-min incubation on ice (to minimize IP hydrolysis), 10-min centrifugation at 15,000 × g, 4°C, and TCA removal through three rounds of supernatant extraction with 2 vol diethyl-ether each. PCA can be removed by equimolar KHCO3 addition (~0.5 M) and centrifugal removal (as above) of the insoluble K-perchlorate. However, the precipitate may contain trapped IP-metal complexes, which form at a pH > 7.0, reducing the IP yield in the supernatant (Lin et al., 2009). Addition of each 20 µg of unlabeled IP3 and IP4 may enhance extraction of these IPs, in particular of IP4 (Wreggett et al., 1987). Replicate samples can be pooled prior to extraction to increase extracted radioactivity.
Poor [3H] or [32P] labeling	Inadvertent cold chase	In 32P-labeling experiments, it is important to avoid inadvertent cold chases by adding stimuli in phosphate-containing solutions (e.g., monoclonal antibodies in hybridoma culture medium). For studies of [3H] inositol lipids, it is recommended that cells be stimulated in the labeling medium (i.e., in the presence of [3H] inositol) to avoid any changes in specific activity during the assay.
Cell activation modulating control compounds have no or little effect	Reduced effective concentration	Reduce FCS in the medium, which may bind compound and reduce its effective concentration. Titrate compound. Change carrier/solvent.
Cell activation modulating control compounds have no or little effect	Compound may not enter cell or undergo fast metabolization/inactivation within the cell	Add detergents such as Pluronic to the compound to improve uptake. Use less polar compound derivatives such as AM/PM esters that are hydrolyzed into the active species by cellular enzymes to improve uptake and, possibly, stability of the compound. Use caged compounds that are taken up and then released very quickly and homogeneously via UV-irradiation, resulting in a compound burst. Or use alternative compounds with improved uptake and stability.
Insufficient stimulation	Stimulation regimen not optimized	Optimize stimulation reagent, its concentration, and application time
Insufficient stimulation	Compromised cell viability/fitness	Improve cell viability/fitness as described above
Insufficient stimulation	High background activity	Rest cells in medium for 15 to 30 min or longer at 25° to 37°C before stimulation
Low HPLC sensitivity for detecting [3H] PIs or IPs	Poor cell viability or labeling	Troubleshoot cell viability and labeling as described above
Low HPLC sensitivity for detecting [3H] PIs or IPs	Low capacity flow cell	Use larger capacity flow cell; however, this may decrease resolution
Low HPLC sensitivity for detecting [3H] PIs or IPs	LSC analysis too insensitive	Use in-line β-detector (see Basic Protocol 2)
Low HPLC sensitivity for detecting [3H] PIs or IPs	Radio-detection too insensitive for given cell amount and labeling efficiency	Use an alternate detection method, such as MDD-HPLC (see Basic Protocol 3 and Alternate Protocol 3; Table 11.1.1).
Low HPLC resolution	Column overloaded or too short	Use larger or longer column (25–cm; Whatman no. 4621-1507). Vary column matrices or separation gradient buffer. HPLC anion exchange matrices, buffers and other aspects of the method are reviewed in detail in Wreggett et al. (1990); Guse et al. (1995b); Singh and Jiang (1995); Williams and Frasca (1998); Kuksis (2003); Azevedo and Saiardi (2006); and Berrie et al. (2007).
Low HPLC resolution	Gradient time too short	Increase gradient time
Low HPLC resolution	Gradient steps too steep	Decrease steepness
Low HPLC resolution	Flow rate too fast	Decrease flow rate
Low HPLC throughput	Number of HPLC columns limiting	Add a second column to run while the first column is re-equilibrated to double throughput
Low HPLC throughput	Gradient time longer than needed	Shorten gradient time, but this may reduce resolution
Low HPLC throughput	Manual sample exchange too slow	Use auto-sampler, which allows automated non-stop running. Neutralized samples are relatively stable for >24 hr at room temperature, allowing long queues.
HPLC resolution deteriorates	Column matrix deteriorates after ~50 runs	Regenerate/re-equilibrate column as described in the respective HPLC protocols and in the respective column manufacturer’s instructions
Insufficient TLC resolution	Suboptimal solvent	Try alternative solvents. For example, 45:35:10 chloroform/methanol/4 N NH4OH (Lapetina and Siess, 1987) allows excellent separation of PI, PIP, and PIP2 but should only be used with [3H] inositol-labeled samples (PI is not well-separated from PA; the latter can be labeled with [32P] but not with [3H] inositol). For additional discussions of TLC, see Singh and Jiang (1995); Hatzack and Rasmussen (1999); Kuksis (2003); Berrie et al. (2007); Otto et al. (2007); Sergeant and McPhail (2007).

^aFor additional HPLC troubleshooting, see Wreggett et al. (1990); Singh and Jiang (1995); Kuksis (2003); Azevedo and Saiardi (2006); and Berrie et al. (2007).