Published customer image: . LYNX Rapid Fluorescein Antibody Conjugation Kit (LNK061) used for conjugation of anti Cyp11a1 antibody to fluoriscein and subsequent use in flow cytometry. Image caption: . The Steroid Synthesis Pathway in Th2 Cells Produces Pregnenolone: Steroid Production In Vitro during Th2 Polarization and In Vivo, in Response to Helminth Infection: (A) Quantitative detection of pregnenolone by LC-MS/MS. Naive Th cells were cultured in Th1 or Th2 activation-differentiation conditions. After 3 days of stimulation, cells were rested for 2 days with equal cell density. Cell supernatants were extracted for steroid profiling. Bars represent the mean pregnenolone concentration SEM. p values were calculated by unpaired two-tailed t test. (B) Quantitative detection of pregnenolone by competitive ELISA. Th cells were stimulated for 3 days and rested for 3 days before supernatants were analyzed by competitive ELISA. Bars represent the mean concentration SEM from four independent experiments. p values were calculated using the unpaired two-tailed t test. (C) Th2 supernatants were analyzed by ELISA for pregnenolone production as described in (B) with or without the presence of Cyp11a1 inhibitor, aminoglutethimide (AG). p values were calculated by unpaired two-tailed t test. Error bars are SDs from the mean. (D) Schematic flowchart of in vivo experimental design. C57BL/6 mice were infected with nematode larvae ( Nippostrongylus brasiliensis ) for 10 days. On day 5 and day 10 postinfection, CD4+ Th cells were purified from spleens and mesenteric lymph nodes (MLN). They were then immediately analyzed by FACS for Cyp11a1 expression, or ex vivo cultured for 24 hr for detection of pregnenolone production by ELISA. (E) Cyp11a1 and GATA3 expression detected by FACS. Data shown were obtained from pooled samples of spleen and MLN of five mice and represent two experiments (n=5 and n=3 for each condition). p values were calculated using the unpaired two-tailed t test using data obtained from individual mice (n=[5 + 3]=8). (F) Pregnenolone concentration of ex vivo cultured Th cell supernatant was measured by quantitative ELISA. Results shown are mean pregnenolone concentration SD from both spleen and MLN. Experiments were performed twice with eight mice per condition. p values were calculated using unpaired two-tailed t test. From: Mahata B, Zhang X, Kolodziejczyk AA, Proserpio V, Haim-Vilmovsky L, Taylor AE, Hebenstreit D, Dingler FA, Moignard V, G ttgens B, Arlt W, McKenzie AN, Teichmann SA. Single-cell RNA sequencing reveals T helper cells synthesizing steroids de novo to contribute to immune homeostasis. Cell Rep. 2014 May 22;7(4):1130-42 .