organism: Mus musculus
description: Protocols: Two classes of CD32NKp46+NK1.1+T-bet+ ILC1s are known to be present in the virgin mouse uterus: CD49a+ trNK- like cells and CD49a2DX5+ cNK-like cells. cNK (conventional NK): Similar to splenic cNK cells, uterine cNK- like cells express Eomes and are termed uterine cNK cells. trNK (tissue-resident NK): In contrast with liver trNK cells, the largest subset in the uterus express Eomes, named Eomes+ uterine trNK cells. Uterine trNK cells are therefore different from Eomes- liver trNK cells but similar to Eomes+ thymic NK cells and salivary gland NK cells. As originally defined in (Doisne et al, 2015; 10.4049/jimmunol.1500689). To investigate further mouse uterus, liver and spleen were processed using a protocol involving both mechanical and enzymatic processing. Finely minced tissues were digested in Accutase (Invitrogen) for 35 minutes at 37 degreeC with gentle orbital agitation (80 rpm) or in Liberase DH (Roche) as described previously (Doisne et al., 2015) for functional assays. Following digestion, tissues were passed through the cell strainer (100 um for uterus, 70 um for liver and 40 um for spleen) using a plunger to mechanically dissociate remaining tissues. Leukocytes were then isolated using 80%/40% Percoll gradient (GE Healthcare Life Sciences) and red blood cell lysis was performed on the resulting cell pellet using BD Pharm Lyse buffer (BD Biosciences) according to manufacturer's instructions. Uterine and liver ILC1 were sorted as live CD45+CD3-veCD19-veCD11blow/-veNK1.1+NKp46+CD49a+Eomes-ve, uterine trNK as live CD45+CD3-veCD19-veCD11blow/-veNK1.1+NKp46+CD49a+Eomes+ and uterine and liver cNK as live CD45+CD3-veCD19-veCD11blow/-veNK1.1+NKp46+CD49a-veEomes+. Samples were sorted on FACSAria III instrument (BD Biosciences). Group 1 ILCs from uterus and liver were sorted into the TRI Reagent (Sigma). RNA extraction was carried out by following TRI Reagent manufacturer's instructions, followed by RNeasy Micro kit (Qiagen). All RNA quality controls and quantifications were performed using RNA 6000 Pico on the 2100 Bioanalyzer instrument (Agilent). Group 1 ILCs' cDNA was amplified using Ovation RNA-seq system V2 (NuGEN) and DNA libraries were produced using Ovation Ultralow System V2 (NuGEN) and following manufacturer's instructions. All mRNA and cDNA quality controls and quantifications were performed using RNA 6000 Pico and High Sensitivity DNA kits (Agilent) on the 2100 Bioanalyzer instrument (Agilent).
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2018-09-01T17:02:14Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2018-05-24T14:03:56Z
attributes.tag: External Id
attributes.value: SAMEA4682738
attributes.tag: INSDC center name
attributes.value: Centre for Trophoblast Research, University of Cambridge
attributes.tag: INSDC first public
attributes.value: 2018-09-01T17:02:14Z
attributes.tag: INSDC last update
attributes.value: 2018-05-24T14:03:56Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-6812:Sample 12
attributes.tag: age
attributes.value: 8 to 12
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell type
attributes.value: group 1 innate lymphoid cell
attributes.tag: common name
attributes.value: house mouse
attributes.tag: genotype
attributes.value: wild type genotype
attributes.tag: organism part
attributes.value: liver
attributes.tag: phenotype
attributes.value: CD45+CD3-veCD19-veCD11blow/-veNK1.1+NKp46+CD49a-veEomes+
attributes.tag: sample name
attributes.value: E-MTAB-6812:Sample 12
attributes.tag: scientific_name
attributes.value: Mus musculus
attributes.tag: strain
attributes.value: C57BL/6