![Figure 6. SAP-deficient murine and hu-iNKT cells form fewer conjugates with antigen-pulsed targets. iNKT cells from B6, tamoxifen-treated Sapfl/flCre– or Sapfl/flCre+ mice and EL4 cells were stained with 250 nM carboxyfluorescein succinimidyl ester and bodipy red succinimdyl ester, respectively. iNKT cells were mixed at a 1:1 ratio with PBS44-loaded or unloaded targets, spun down at 1200 rpm for 2 minutes, and incubated at 37°C for varying times. Conjugate frequencies were analyzed by flow cytometry for the presence of cellular aggregates staining positive for bodipy red succinimdyl ester and carboxyfluorescein succinimidyl ester. (A-B) Mean percent iNKT cells (of total iNKT cells) in conjugate with PBS44-loaded or unloaded EL4 cells. (C-D) Mean percent of SAP+ or SAP− iNKT cells ± SEM in conjugate with PBS44-loaded EL4 cells. iNKT cells were flow-sorted from B6 mice (A), tamoxifen-treated Sapfl/flCre− (SAP+) or Sapfl/flCre+ (SAP−) mice (C), or control (SAP+) or SAP-specific (SAP−) siRNA transfected hu-iNKT cells (D). Percent iNKT cells in conjugate was calculated by using the formula [% iNKT-containing conjugates (upper right quadrant)]/[% iNKT-containing conjugates (upper right quadrant) + % iNKT cells not in conjugate (lower right quadrant)] × 100. Data presented are from 3 experiments. Statistical significance was determined by unpaired two-tailed t test. **P < .001.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/121/17/10.1182_blood-2012-11-468868/4/3386f6.jpeg?Expires=1722779198&Signature=ycFprLQpJvEFC~BPLwQNGYU~QoAfG38oY4rQ-6RqRC6H4lG2IrzMpKN8PB50tzN5xTQN7IyIRR7iqzigXl4HaGh4M66IzaU8JtIYhZ7quuX0URcmP72tNbfSTVv05jIRZYo-n~TtovKfVZ8mDNQwVeh5wy4I46WVQ0gGdTbK1eTeJXJcNshlFZNP89mxh4rNB227ZXvPGElTAirH2x4bJEA~Ri7hH3scDPUwtMbMdZryq-I-ttVMzX0jlQhhjoRRMiljPECLf1H29EBpveR41wDr4Xn0wWPSeBC7PAApWC~fH1eyGLXwtqh6eAb17ECtH1a1dvXw96vU2DSZqIckKA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
SAP-deficient murine and hu-iNKT cells form fewer conjugates with antigen-pulsed targets. iNKT cells from B6, tamoxifen-treated Sapfl/flCre– or Sapfl/flCre+ mice and EL4 cells were stained with 250 nM carboxyfluorescein succinimidyl ester and bodipy red succinimdyl ester, respectively. iNKT cells were mixed at a 1:1 ratio with PBS44-loaded or unloaded targets, spun down at 1200 rpm for 2 minutes, and incubated at 37°C for varying times. Conjugate frequencies were analyzed by flow cytometry for the presence of cellular aggregates staining positive for bodipy red succinimdyl ester and carboxyfluorescein succinimidyl ester. (A-B) Mean percent iNKT cells (of total iNKT cells) in conjugate with PBS44-loaded or unloaded EL4 cells. (C-D) Mean percent of SAP+ or SAP− iNKT cells ± SEM in conjugate with PBS44-loaded EL4 cells. iNKT cells were flow-sorted from B6 mice (A), tamoxifen-treated Sapfl/flCre− (SAP+) or Sapfl/flCre+ (SAP−) mice (C), or control (SAP+) or SAP-specific (SAP−) siRNA transfected hu-iNKT cells (D). Percent iNKT cells in conjugate was calculated by using the formula [% iNKT-containing conjugates (upper right quadrant)]/[% iNKT-containing conjugates (upper right quadrant) + % iNKT cells not in conjugate (lower right quadrant)] × 100. Data presented are from 3 experiments. Statistical significance was determined by unpaired two-tailed t test. **P < .001.
