To avoid depleting Ly6C+ NK cells we developed a custom antibody cocktail as follows: splenocytes were labeled with 10 μg per spleen of biotin conjugated antibodies against CD3 (17A2), CD19 (6D5), CD8 (53-6.7), CD88 (20/70), Ly6G (1A8), SiglecF (S17007L), TCRβ (H57-597), CD20 (SA275A11), CD172a (P84) and magnetically depleted from total splenocyte suspensions with the use of anti-biotin coupled magnetic beads (Biolegend). Splenic single cell suspensions were lysed in red blood cell lysis buffer and resuspended in EasySep™ buffer (Stemcell). Mouse spleens, livers, lungs, lymph nodes, and blood were harvested and prepared into single cell suspensions as described previously11. Mixed bone marrow chimeric (mBMC) mice were generated by depleting host CD45.1xCD45.2 mice by intraperitoneal (i.p.) injection of busulfan (1mg/mL) at 20mg/kg for 3 consecutive days, which were then reconstituted 24 hours later with various mixtures of bone marrow cells from WT (CD45.1) and knockout (CD45.2) donor mice. Thus, to control for the number of UTX copies, experiments were conducted using 6-8 week old age-matched female UTXNKD and WT littermates in accordance with approved institutional protocols. UTX is located on the X chromosome and escapes inactivation, it is expressed from 2 chromosomal copies in females but only one in males. The following mouse strains were used in this study: C57BL/6 (CD45.2) (Jackson Labs, #000664), B6.SJL (CD45.1) (Jackson Labs, #002114), Klra8-/- (Ly49H-deficient), Rosa26ERT2Cre, Ncr1Cre, Utxfl/fl, UTX demethylase dead (UTXDMD) which harbors two point mutations at H1146A and E1148A. Mice were bred at UCLA in accordance with the guidelines of the institutional Animal Care and Use Committee (IACUC). Mice were monitored and weighed daily and sacrificed when body weight dropped over 20% from initial weight. ![]() ![]() injection of 7.5 x 103 plaque-forming units (PFU) in 0.5 mL of PBS. Experimental mice in studies were infected with MCMV by i.p. ![]() MCMV (Smith) was serially passaged through BALB/c hosts three times, and then salivary gland viral stocks were prepared with a dounce homogenizer for dissociating the salivary glands of infected mice 3 weeks after infection. GEO help: Mouse over screen elements for information.
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