cat no | io1102 Early Access
CRISPRa-Ready ioMicroglia Male
Male human iPSC donor-derived microglia expressing modified dCas9 for gene activations and CRISPRa screens
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Cryopreserved human iPSC-derived cells powered by opti-ox that are ready for experiments within days
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Microglia constitutively expressing modified dCas9 optimised for gene activations and CRISPRa screens
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Perform key phagocytic and cytokine secretion functions, and are co-culture compatible
Male human iPSC donor-derived microglia expressing modified dCas9 for gene activations and CRISPRa screens
Flow cytometry analysis demonstrates gene activation of P2RY12 upon lentiviral gRNA delivery
Flow cytometry analysis confirmed robust P2RY12 gene activation in CRISPRa-Ready ioMicroglia following lentiviral delivery of a P2RY12-targeting gRNA on day 10 post-thaw using VPx-VLP for enhanced lentiviral gRNA delivery. Gene activation was assessed 5 days later.
(A) 41% of cells were GFP+ indicating successful delivery of the P2RY12-targeting gRNA, with 83% of these GFP+ cells showing robust activation.
(B) The dCas9 transcriptional activator induced a 10.3-fold increase in P2RY12 protein expression (orange) relative to non-targeting control (grey), as measured by geometric mean fluorescence intensity (GMFI) in the GFP+ population.
Immunocytochemistry analysis demonstrates gene activation of SOX11 upon lentiviral gRNA delivery in a 96-well plate
SOX11-targeting gRNAs were delivered to CRISPRa-Ready ioMicroglia on day 10 post-thaw via lentiviral transduction in a 96-well plate format, using VPx-VLP for enhanced lentiviral gRNA delivery. After 5 days, transduction and SOX11 activation efficiencies were quantified by immunocytochemistry. Comparable results were observed in 24-well plates.
(A) Representative images showing activation of SOX11 in a majority of transduced (GFP+) cells.
(B) Quantification of GFP+ cells reveals 20-30% transduction efficiency.
(C) A high SOX11 activation efficiency of 80% was achieved in cells transduced with SOX11 gRNA.
CRISPRa-Ready ioMicroglia show ramified morphology by day 4
CRISPRa-Ready ioMicroglia mature rapidly; key ramified morphology can be identified by day 4 and continues through to day 10, similar to ioMicroglia Male (io1021). Day 1 to 10 post-thawing; 100x magnification.
CRISPRa-Ready ioMicroglia homogeneously express IBA1
Immunofluorescent staining on day 10 post-revival demonstrates homogenous expression of key microglia marker IBA1 and ramified morphology in CRISPRa-Ready ioMicroglia, similar to ioMicroglia Male (io1021). 100x magnification.
Phagocytosis of E. coli particles by CRISPRa-Ready ioMicroglia
Phagocytosis assay using pHrodo E. coli BioParticles at day 10 post-thaw demonstrates efficient uptake of bioparticles by CRISPRa-Ready ioMicroglia, in a similar manner to ioMicroglia Male (io1021) over 24 h. The graphs display the proportion of cells phagocytosing (left), and the fluorescence intensity per cell displaying degree of phagocytosis (right). The addition of Cytochalasin D (CytoD), an inhibitor of actin polymerisation, significantly decreased E.coli particle uptake as expected.
CRISPRa-Ready ioMicroglia secrete pro-inflammatory cytokines upon activation
CRISPRa-Ready ioMicroglia were stimulated at day 10 post-thaw with LPS 100 ng/mL and IFNɣ 20 ng/mL for 24 hours. Cell culture supernatants were collected and cytokine secretion levels were quantified by ELISA. Upon activation, these cells secreted TNF-α and IL-6 at comparable levels to wild-type ioMicroglia Male (io1021).
View the cytokine secretion protocol used to generate this data.
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Emmanouil Metzakopian
