negative selection direct lineage cell depletion kit  (Miltenyi Biotec)

Journal: bioRxiv

Article Title: The epigenetic reader MORC3 is required for T cell development in the thymus

doi: 10.1101/2025.03.05.641591

Figure Lengend Snippet: (A) Schematic representation of MORC3 protein, its different domains and the MommeD41 mutation. (B) Western blot showing MORC3 protein levels in E14.5 fetal liver and E18.5 spleen and thymus, two Morc3 +/+ biological replicates. VINCULIN was used as a loading control. (C) Western blot showing MORC3 protein levels in E18.5 thymus, two biological replicates per genotype. VINCULIN was used as a loading control. (D) (top) Representative macroscopic pictures of thymus from E18.5 Morc3 +/+ , Morc3 +/MD41 and Morc3 MD41/MD41 littermates (scale bars, 0,5cm). (bottom) Total cell count of E18.5 thymus from Morc3 +/+ , Morc3 +/MD41 and Morc3 MD41/MD41 littermates (n = 8 Morc3 +/+ , n = 8 Morc3 +/MD41 , n = 5 Morc3 MD41/MD41 ). Data are presented as mean with dot, square or triangle as individual values; error bar represents standard deviation (SD). (E) Representative flowcytometry plots of T cell subsets in Morc3 +/+ , Morc3 +/MD41 and Morc3 MD41/MD41 E18.5 thymus. CD4 and CD8 were used as markers to define DN, DP and single positive CD4+ and CD8+. (F) (left) Bar plot depicting the percentage of CD4+CD8+ Double Positive (DP) within CD45.1+ cells (pre-gated on SSC-A/FSC-A, single, live, Lin-) and (right) total cell number in E18.5 thymus (n = 8 Morc3 +/+ , n = 8 Morc3 +/MD41 , n = 5 Morc3 MD41/MD41 , 3 independent experiments). Data are presented as mean with dot, square or triangle as individual values; error bar represents standard deviation (SD). (G) (left) Bar plot showing the percentage of CD4-CD8-Double Negative (DN) within CD45.1+ cells (pre-gated on live, Lin-, CD3-) and (right) total cell number in E18.5 thymus (n = 8 Morc3 +/+ , n = 8 Morc3 +/MD41 , n = 5 Morc3 MD41/MD41 , 3 independent experiments). Data are presented as mean with dot, square or triangle as individual values; error bar represents standard deviation (SD). For panels D, F and G statistical significance was determined using two-sided t test. (H) Representative flowcytometry plots of DN cells in Morc3 +/+ , Morc3 +/MD41 and Morc3 MD41/MD41 E18.5 thymus. In the left column, CD25 and CD44 were used as markers to define DN1, DN2, DN3, DN4 T cell subsets within CD4-CD8-CD3-cells. In the middle column, c-kit was used as marker to define ETP and DN1 within CD44+CD25-cells. In the right column, c-kit was used as marker to define DN2a and DN2b within DN2 cells. (I) (top) Bar plot showing the percentage of DN cells in CD45.1+ cells (pre-gated on SSC-A/FSC-A, single, live, Lin-) and (bottom) total cell number in E18.5 thymus. (n = 8 Morc3 +/+ , n = 8 Morc3 +/MD41 , n = 5 Morc3 MD41/MD41 , 3 independent experiments). Data are presented as mean with dot, square or triangle as individual values; error bar represents standard deviation (SD). Statistical significance was determined using two-way ANOVA for multiple comparisons. For all figures: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001.

Article Snippet: E14.5 fetal liver-derived lineage negative cells were transduced using RetroNectin (T100B, Takara Bio Inc.) coated wells following the manufacturer’s instructions.

Techniques: Mutagenesis, Western Blot, Control, Cell Counting, Standard Deviation, Marker

Journal: bioRxiv

Article Title: The epigenetic reader MORC3 is required for T cell development in the thymus

doi: 10.1101/2025.03.05.641591

Figure Lengend Snippet: (A) Schematic illustration of the differentiation of T cells from E14.5 fetal liver-derived lineage negative (Lin- : CD3-, CD4-, B220-, Gr1-, NK1.1-, Cd11c-, TER119-) cells co-cultured on OP9-DL1 cells. (B) Representative flowcytometry plots of Morc3 +/+ and Morc3 MD41/MD41 DN1, DN2, DN3 and DN4 cells generated from E14.5 fetal liver-derived lineage negative cells co-cultured on OP9-DL1 cells for 7 days. (C) (top) Percentage of DN1, DN2, DN3 and DN4 cells within Thy1.1+ cells (pre-gated on live, Lin-, CD45.1+) and (bottom) total cell number (n = 5 biological replicates per genotype, 3 independent experiments); data are presented as mean with dots/triangles as individual values; error bar represents SD. Statistical significance was determined using two-sided t test. (D) RT-qPCR analysis of relative Morc3 , Bcl11a , Spi1 and Tcf7 mRNA levels in Morc3 +/+ and Morc3 MD41/MD41 sorted DN1 cells after 7 days of co-culture (normalized to β-actin; n = 4 biological replicates; data are presented as mean with dots/triangles as individual values; error bar represents SD. Statistical significance was determined using two-sided t test. (E) Representative flowcytometry plots of Morc3 +/+ and Morc3 MD41/MD41 myeloid (Gr1+, Cd11b+), NK (NK1.1+, Gr1-,Cd11b-) and B cells (CD19+, B220+, Gr1-,Cd11b-, NK1.1-, Thy1.1-) generated from E14.5 fetal liver-derived lineage negative cells co-cultured on OP9-DL1 cells for 7 days. All cells were pre-gated on single, live, CD45.1+. (F) (top) Percentage of myeloid, NK and B cells within CD45.1+ cells (pre-gated on single, live) and (bottom) total cell number (n = 4 biological replicates/genotype, 2 independent experiments); data are presented as mean with dots/triangle as individual values; error bar represents SD. Statistical significance was determined using two-sided t test. For all figures: *P≤0.05, **P≤0.01, ***P≤0.001, ****P ≤ 0.0001

Article Snippet: E14.5 fetal liver-derived lineage negative cells were transduced using RetroNectin (T100B, Takara Bio Inc.) coated wells following the manufacturer’s instructions.

Techniques: Derivative Assay, Cell Culture, Generated, Quantitative RT-PCR, Co-Culture Assay