Enrichment of Nkx2-2enh::CD14 Cells

The Nkx2-2enh::CD14 transgene-expressing cell fraction was enriched by MACS^{29 (link)} from in vitro differentiation cultures. Developing embryoid bodies were dissociated with Accumax at 37 °C at 400 rpm shaking for 10 min. Cells were labelled with anti-CD14-APC primary at 1:50 concentration (Supplementary Table S3), and after a wash, mouse anti-APC MicroBeads (130-090-855, Miltenyi) at 1:5 concentration in MACS buffer for 15 min each at 4 °C in a Miltenyi Biotec MACSmix Tube Rotator. Pre-sort, flow through and eluate fractions were analysed using a BD Fluorescence-activated cell sorting (FACS) Canto II machine (NIHR Guy’s and St. Thomas’ Biomedical Research Centre). DAPI was added at 1 µg/ml to exclude the dead cells just before the analysis. 10,000 cells were analysed using the FlowJo software (BD Biosciences). Live single cells were gated based on forward and side scatter (FSC and SSC) parameters, as well as DAPI absorbance. Unstained cells were used to gate for an APC-positive fraction.

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Berzanskyte I., Riccio F., Machado C.B., Bradbury E.J, & Lieberam I. (2023). Enrichment of human embryonic stem cell-derived V3 interneurons using an Nkx2-2 gene-specific reporter. Scientific Reports, 13, 2008.

Publication 2023

MACSmix Tube Rotator

Buffer Cells Dapi Embryoid bodies Microbeads Mouse Transgene

Corresponding Organization : King's College London

Top 5 similar protocols

Variable analysis

independent variables

Cell enrichment by MACS from in vitro differentiation cultures

dependent variables

Fraction of Nkx2-2enh::CD14 transgene-expressing cells

control variables

Dissociation of developing embryoid bodies with Accumax at 37 °C, 400 rpm shaking for 10 min
Labeling of cells with anti-CD14-APC primary antibody at 1:50 concentration
Labeling of cells with mouse anti-APC MicroBeads at 1:5 concentration in MACS buffer for 15 min at 4 °C
Analysis of pre-sort, flow through and eluate fractions using a BD Fluorescence-activated cell sorting (FACS) Canto II machine
Addition of DAPI at 1 µg/ml to exclude dead cells
Analysis of 10,000 cells using the FlowJo software
Gating of live single cells based on forward and side scatter (FSC and SSC) parameters, as well as DAPI absorbance
Use of unstained cells to gate for an APC-positive fraction

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