Additional file 2 of Neutrophils cultured ex vivo from CD34+ stem cells are immature and genetically tractable
Creators: name: Naveh, Claire A.
name: Roberts, Kiran
name: Zakrzewski, Przemysław
name: Rice, Christopher M.
name: Ponce-Garcia, Fernando M.
name: Fleming, Kathryn
name: Thompson, Megan
name: Panyapiean, Nawamin
name: Jiang, Huan
name: Diezmann, Stephanie
name: Moura, Pedro L.
name: Toye, Ashley M.
name: Amulic, Borko
Publisher: name: figshare
Year: 2024
Subjects: subject: Space Science
subject: Medicine
subject: Microbiology
subject: FOS: Biological sciences
subject: Cell Biology
subject: Genetics
subject: Physiology
subject: Immunology
subject: FOS: Clinical medicine
subject: Developmental Biology
subject: Hematology
subject: Infectious Diseases
subject: FOS: Health sciences

description: Supplementary material 2 : Fig. S1 Additional characterisation of cultured neutrophils. A: Schematic outlining culture procedure (top) or acquisition of native neutrophils. Figure generated in Biorender. B: Representative gating strategy of native and cultured cells. Native cells were gated on a singlet, appropriately sized population where monocytes and eosinophils were gated out using CD14 and IL-5R expression respectively. Cultured neutrophils were gated on a singlet, appropriately sized, live population using granulocyte markers CD66b and CD15. C: Quantitative description of nucleation in cultured neutrophils at day 17 of differentiation, n=3. D: Percentage of culture classified as CD14high (monocytes) and CD14low (neutrophils) by flow cytometry at day 17 of differentiation, n=3. E: CD14 MFI measured by flow cytometry at day 17 of culture, n=3. F: Analysis of viability by flow cytometry at days 17-21 of differentiation, live= annexin V-; PI -; dead: annexin V+; PI+, n=1. G: Percent of cells expressing CD66B, measured by flow cytometry, at late stage of the culture protocol, n=2-3. Fig. S2 Native and GCSF-D neutrophil staining properties and purity A: Representative scatter plots of neutrophils gated on size, CD45, CD15 and CD66b expression. B-C: CD101 (B) and CD10 (C) expression in peripheral blood neutrophils from native (red), GCSF-D (blue) and cultured neutrophils (black), with fluorescence minus one (FMO) controls for cultured neutrophils and peripheral blood neutrophils displayed in grey. D: Percentage of cells expressing CD10, in native, GCSF-D and cultured (day 17) neutrophils, n=3-4. E-G: Representative FACS plots of CD15 (F), CD66B (G) and CD11b (H), including unstained controls. H: Representative images of NETs induced by 10 uM A23187, stained with SYOTO Green and SYTOX Orange. I: Quantification of I, n=2-3. Fig. S3 Cultured neutrophils differ in mitochondrial and granule protein abundance A: Representative sorting gates for native and cultured neutrophils before TMT mass spectrometry. B: Top 8 unique Reactome 2022 pathway terms of enriched (red) and under-represented (blue) genes using the GO Biological Pathway module. C: Normalised protein abundances of key TCA cycle and respiratory electron transport proteins, n=3 differentiations. D: Normalised abundances of key granule proteins quantified between cultured and primary neutrophils in the dataset reported in this work (n=3, Naveh C et al.) and between GCSF-treated neutrophils and neutrophils from healthy donors (n = 3, data from Aymonnier K et al. 2024). An inter-dataset comparison (comparing Log2FC of both datasets) is highlighted above the data; intra-dataset comparisons against baseline are highlighted below the data. ns = no significant difference; * = P &lt; 0.05; ** = P &lt; 0.01. E-F: Enriched (E) and under-represented (F) proteins processed using the STRING database using a medium confidence setting to produce protein-protein interaction networks. Clusters are shown in different colours: E) ribosomal proteins in red, mitochondrial proteins in blue and granule proteins in green and in F) innate immune system and degranulation in red, mRNA processing in blue and chromatin organisation in green. Fig. S4 CD66b expression and viability is unchanged by CRISPR/Cas9 mediated knockout in cultured neutrophils A: Percentage of PI positive cells in Scr vs β2M (left) and CD11b (right) KO cells respectively by flow cytometric surface marker staining on day 7 of culture. B: Percentage of CD66b expressing cells in β2M (left) and CD11b (right) CRISPR/Cas9 KO cells, by flow cytometric surface marker staining on day 17 of culture. Table 1: Proteins uniquely expressed in cultured neutrophils and not native neutrophils. Table 2: Antibodies and fluorescent dyes used for flow cytometry Table 3: Synthego guide RNA for CRISPR-Cas9 gene editing