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. Author manuscript; available in PMC: 2014 Mar 1.
Published in final edited form as: J Immunol. 2013 Jan 28;190(5):2058–2067. doi: 10.4049/jimmunol.1201503

Figure 3. shRNA-mediated silencing of SphK2, but not SphK1, results in impaired degranulation and calcium responses in PDMC and BMMC.

Figure 3

Lentivirus transduced PDMC (A, B) or BMMC (C, D) were sensitized with anti-DNP IgE as described in methods. Cells were washed and then stimulated with the indicated concentrations of antigen. (A, C) Degranulation was calculated as the percentage of β-hexosaminidase released to the incubation media as compared to the total β-hexosaminidase cellular content. Data shown are the average of 3-5 independent cultures done in triplicate ± S.E. Statistical significance compared to nontarget control, * P<0.05, **P<0.01. (B, D) Lentivirus transduced cells were loaded with Fluo4-AM and FuraRed-AM. Following antigen (Ag) stimulation, calcium responses were measured using confocal microscopy and are shown as a ratio of intensity of Fluo4-AM/FuraRed-AM. One representative experiment is shown averaging 15 to 20 individual cells/ time point. Data shown are a mean ± S.E. Similar results were obtained in 3 independent cultures measuring calcium as the ratio of emission at 510 nm when FURA-2AM loaded cells were excited at 340 and 380 nm in populational analysis (data not shown).