TABLE 1.

Troubleshooting table.

Step	Problem	Possible reason	Solution
2, 24, 27	Low RV transduction efficiency	Insufficient activation of CD8+ T cells	Check cell activation by flow cytometry and optimize culture conditions, if needed: Time: at least 18 h is needed for CD8+ T cells to become activated for optimal RV transduction Reagents: the batch of serum used in cell culture is critical. Use fresh and sterile antibodies that were divided into aliquots upon arrival. Use a fresh aliquot of IL-2 and add 2-mercaptoethanol to complete RPMI-1640 to maintain IL-2 activity Cell density: a concentration of at least 1 × 106 cells per ml during in vitro stimulation is recommended
30, 52	Low RV transduction efficiency	The temperature was not optimal during the procedure	Cells should be kept above room temperature to maintain activity. Do not place cells on ice at any time. Set the centrifuge to 30 °C for spin-transduction
56, 58	Low RV transduction efficiency	Low viability of T cells during transduction	Excessive exposure to Polybrene: lower the concentration or shorten the time of exposure to viral supernatant. Consider switching to RetroNectin if use of Polybrene continues to result in low viability
47	Low RV transduction efficiency	Residual Percoll particles in cell suspension	Wash the cells at least twice with complete RPMI-1640 before RV transduction
55	Low RV transduction efficiency	The wrong RV system was used	Use an ecotropic RV system for standard mouse T-cell experiments
55	Low RV transduction efficiency	The RV stock titer was too low	Check the RV titer before starting in vitro T-cell stimulation. Use of high-titer stock (>107 reporter-positive focus-forming units/ml) is recommended
56	Low RV transduction efficiency	The wrong tissue-culture plates were used for spin-transduction and incubation	Noncoated plates should be used for spin-transduction
1	No visible interface layer or low cell recovery after Percoll separation	The input cell number used was too low	An input cell number of at least 5 × 106 is recommended. Increase the number of spleens if necessary
30, 42	No visible interface layer or low cell recovery after Percoll separation	The temperature of the Percoll reagents and centrifugation were not optimal	Leave the Percoll reagents at room temperature for at least 1 h or use a water bath. Make sure that the temperature during centrifugation is set to 25 °C
40, 42	No visible interface layer or low cell recovery after Percoll separation	The 30 and 60% (vol/vol) Percoll layers were disturbed	Care must be taken when underlaying 60% (vol/vol) Percoll and moving the tubes to/from the centrifuge. Accelerator and brake should be off during centrifuge
30, 34	No visible interface layer or low cell recovery after Percoll separation	30 and/or 60% (vol/vol) Percoll solutions were not appropriately prepared	Check the reagents. Consider using a density marker if refreshing all reagents fails to solve the problem
34	No visible interface layer or low cell recovery after Percoll separation	The combination of 30 and 60% (vol/vol) Percoll is not optimal in the experimental setting	Test whether modifying the Percoll concentration by 5% increments works better
1	Low levels of RV-transduced T cells in vivo	Rejection of transferred T cells due to unmatched genetic background	Check the genetic background of the donor mouse and run a test transfer experiment without RV transduction
55	Low levels of RV-transduced T cells in vivo	Rejection due to the RV marker	Compare with different RV marker(s) in individual experimental settings. Consider switching RV marker if rejection is suspected
67	Low levels of RV-transduced T cells in vivo	The number of transferred T cells was too low	Titrate the optimal cell number for transfer in individual experimental settings
68	Low levels of RV-transduced T cells in vivo	Low RV transduction efficiency	Check the RV transduction efficiency on day 2 in vitro
73	Low levels of RV-transduced T cells in vivo	Loss of RV-transduced T cells by Histopaque centrifugation during sample preparation	Switch to RBC lysis buffer when preparing single-cell suspension during acute phase
74	Low levels of RV-transduced T cells in vivo	Leakage of RV-derived fluorescent marker(s) in intracellular staining application	Add a pre-fixation step with 2% (vol/vol) PFA in PBS between staining surface molecules and permeabilizing steps