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Blood, Vol. 112, Issue 12, 4591-4597, December 1, 2008
Suppression of LPS-induced Interferon- and nitric oxide in splenic lymphocytes by select estrogen-regulated microRNAs: a novel mechanism of immune modulation
Blood Dai et al.
112: 4591
Supplemental materials for: Dai et al
Cell separation
Splenic macrophages and T cells were purified by positive selection using mouse CD11b and CD90.2 (Thy1.2) microbeads (Miltenyi Biotec), respectively following the manufacturer’s instructions. Briefly, splenic lymphocytes from estrogen-treated mice were first mixed with CD11b microbeads to isolate macrophages. The negative eluted cells (macrophage depleted splenic lymphocytes) were recounted and were subsequently mixed with CD90.2 microbeads to isolate T cells. The purity of macrophages (around 98%) and T cells (around 82%) were checked by staining the isolated cells with APC-conjugated CD11b and FITC-conjugated CD90.2 (Thy1.2) antibodies, respectively, and analyzed by flow cytometry using the BD FACSARIA in the Flow Cytometry Laboratory at the VMRCVM, Virginia Tech. The cells were adjusted to 5 × 106/ml, plated in 96 well plates, and stimulated with LPS (at 500 ng/ml final concentration) for the indicated time. The supernatants were collected for IFNγ ELISAs and Griess assays, respectively.
Files in this Data Supplement:
- Figure S1. The early induction of LPS-induced IFNγ and LPS-induced nitric oxide in splenic lymphocytes from estrogen-treated mice is dependent on interactions between macrophages and T cells (JPG, 81 KB)
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Whole splenic lymphocytes, purified splenic macrophages, T cells, macrophage-depleted cells, and final negative eluted cells (macrophages and T cell-depleted splenic lymphocytes) were adjusted to 5 × 106 /ml, plated in 96 well plates, and then stimulated with LPS (500 ng/ml at final concentration) for 6 hrs (A), 48 hrs (B). Aliquots of splenic lymphocytes were left unstimulated (medium) and served as baseline controls. To determine the importance of interactions between macrophages and T cells, equal volumes of purified macrophages and T cells were plated in the same well, and then stimulated with LPS. (A) The induction of IFNγ in culture supernatants was determined with ELISAs. (B) The production of nitric oxide in culture supernatants was determined with Griess assays. The graphs show means ± SEM (n ≥ 4 each).
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