Astrios eq cell sorter

Macrophages were flow-sorted from age and sex-matched Cx3cr1^GFP/+ reporter mice. The gating strategy, as illustrated in the figures, guided the precise selection of IMs and AMs. Each experimental time point involved the use of a pair of age and sex-matched mice, ensuring a balanced representation with one male and one female mouse. The flow sorting procedure was conducted employing an Astrios EQ cell sorter (Beckman Coulter Life Sciences) at the CUAMC Cancer Center Flow Cytometry Shared Resource. Following the flow sorting, the isolated cells were collected in a specialized medium (HBSS-Gibco with 2.5% FBS), subjected to centrifugation, accurately counted, and prepared for subsequent sequencing analysis. This systematic approach ensured the acquisition of high-quality data from the sorted IMs and AMs, contributing to the comprehensive understanding of their transcriptional profiles across different conditions.

Transfected HCT116 cells were assayed by flow cytometry after 72-h puromycin selection (96 h after transfection). For flow cytometry collection of the HER2-negative cell population, cells were harvested from two independent biological replicates, washed once in PBS, and resuspended in 1% bovine serum albumin (BSA) in PBS. After adding 3 µl APC-conjugated antihuman CD340 (erbB2/HER-2) antibody (Biolegend #324408), cells were incubated for 30 min at 4 °C. Labeled cells were washed once and resuspended in 1% BSA in PBS. Cell sorting was performed using an Astrios EQ cell sorter (Beckman Coulter) at the UC Davis Flow Cytometry Shared Resource Core. Untreated HCT116 control cells were used to determine the APC signal for HER2-expressing cells, while unlabeled cells were used to determine the sorting gate for HER2-negative cells. Four days after combinatorial epi-dCas9 transfections, HER2-negative cells were collected and replated using standard media. Cells were harvested at indicated time points, dependent on cells reaching ~ 80% confluency. Cells were expanded in 24-well dishes for RNA isolation (5 days, 14 days, 23 days, 39 days, 44 days and 50 days) and in 6-well dishes for DNA methylation analysis (10 days, 17 days and 24 days) and Western blot analysis (40 days). For ChIP assays (24 days), cells were plated in 10-cm dishes.

ADAR3 mutant library covering the top 11 mutations was transformed to S. cerevisiae INVSc1 strain already harboring the yeGFP reporter using a high efficiency lithium protocol (28 (link)). A small fraction of transformation mixture was plated on dropout plate to calculate transformation efficiency. The transformation was estimated to cover at least 50-fold of the theoretical diversity of the library. The remaining transformation mixture was used to inoculate 10 ml CM – ura – trp + 2% glucose media and grew until OD₆₀₀ reaches to 7–8. The glucose culture was then used to inoculate CM – ura – trp + 3% glycerol + 2% lactate media (0.25 ml to 25 ml) and after an additional 36 h of growth (OD₆₀₀ = 1–2), galactose was added to a final concentration of 3% for induction. Cells collected after 24 h induction were washed twice and resuspended with PBS to 20 000 cells/μl. Cells were sorted using Beckman Coulter Astrios EQ cell sorter at UC Davis flow cytometry shared resource laboratory. GFP excitation was at 488 nm and emission at 529/28 nm.

Labeling of cortical neuronal progenitors with CFSEs was achieved as described elsewhere (Govindan et al., 2018 (link)). Briefly, 1 µl of a 5 mM solution of CellTrace CFSE (from the CellTrace CFSE Cell Proliferation Kit, Invitrogen, C34554) and 0.01% Fast Green in DMSO was injected into the third ventricle of E13.5 control and Emx1-NICD embryos. Dams were allowed to recover and injected embryos were collected 1 h post-injection. Embryonic cortices were dissected individually and dissociated into single cells using Papain Dissociation System (Worthington Biochemical, LK003150) following the manufacturer's protocol. Cells were resuspended in FACS media (DMEM/F12 without phenol red supplemented with 10% fetal bovine serum and B-27) and sorted using a Beckman Coulter Astrios EQ Cell Sorter.