CD45.1 BMDCs were incubated with nociceptors from CD45.2 mice or alone and treated with appropriate stimuli. After 8 hours, the cultured cells were harvested, stained, and sorted on a MoFlo Astrios EQ FACS-sorter (Beckman). cDC1s were identified as CD45.1+XCR-1+CD11b−, cDC2s as CD45.1+CD11b+XCR-1−, and pDCs as CD45.1+XCR-1−CD11b−PDCA1+. One thousand cells of each population were sorted directly into 5 μl of lysis buffer (TCL Buffer (Qiagen) with 1% β-mercaptoethanol) and Smart-seq2 libraries were prepared as previously described (74 (link), 75 (link)) with slight modifications. Briefly, total RNA was captured and purified on RNAClean XP beads (Beckman Coulter). Polyadenylated mRNA was then selected using an anchored oligo(dT) primer (5′-AAGCAGTGGTATCAACGCAGAGTACT30VN-3′) and converted to cDNA via reverse transcription. First strand cDNA was subjected to limited PCR amplification followed by transposon based fragmentation using the Nextera XT DNA Library Preparation Kit (Illumina). Samples were PCR amplified for 18 cycles using barcoded primers such that each sample carried a specific combination of eight base Illumina P5 and P7 barcodes and pooled together prior to sequencing. Paired-end sequencing was performed on an Illumina NextSeq500 using 2×25 bp reads.
Moflo astrios eq facs sorter
Manufactured by Beckman Coulter
The MoFlo Astrios EQ FACS-sorter is a high-performance cell sorting instrument designed for flow cytometry applications. It provides precise and efficient cell sorting capabilities without extrapolation on intended use.
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Lab products found in correlation
Nextseq 500, by Illumina (1 mentions)
Tcl buffer, by Qiagen (1 mentions)
Nextera xt dna library preparation kit, by Illumina (1 mentions)
Rnaclean xp beads, by Beckman Coulter (1 mentions)
Collagenase type xi, by Merck Group (1 mentions)
Dispase 2, by Thermo Fisher Scientific (1 mentions)
Chromium single cell reagent kit, by 10x Genomics (1 mentions)
Matrigel, by BD (1 mentions)
Epicult b medium, by STEMCELL (1 mentions)
3 protocols using moflo astrios eq facs sorter
1
Single-cell RNA-seq of DC subsets
2
Single-Cell RNA-Seq of Embryonic Cells
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E14 MKO/tdT and Het/tdT embryos were collected, decapitated and eviscerated. To generate single cells, truncated embryos were incubated in 0.2% (wt/vol) collagenase‐type XI (Sigma‐Aldrich) and 2.4 U mL−1 dispase II (Invitrogen) in DMEM at 37 ℃ 30 min, followed by filtering through a 40‐µm cell strainer. Cells were then centrifuged, resuspended in cold 5% FBS in PBS, and immediately sorted with a Beckman moflo Astrios EQ FACS sorter. Endogenous tdT signal was detected through PE channel and gates were set up according to the positive and negative controls. Sorted tdT+ cells were resuspended in 20% FBS/PBS buffer and loaded onto a droplet‐based library prep platform Chromium (10x Genomics) with a Chromium Single Cell Reagent Kit (version 3) according to the manuscript's instructions. Sequencing was performed on an Illumina nova 6000 and 150‐base pair paired‐end reads. The sequencing and bioinformatics analysis were performed by OE Biotech Co., Ltd. (Shanghai, China). All statistical analyses, unless otherwise specified, were conducted using R.[32 ]
3
Isolation and Characterization of Mammary Stem Cells
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Isolation of mammary epithelial subpopulations was performed as described previously [57] (link). Purified mammary epithelial cells were stained with biotin-conjugated anti-CD31 (catalogue no. 553371; BD) and biotinylated CD45 + and Ter119 + (StemSep murine chimaera cocktail, catalogue no. 13058C; Stem Cell Technologies) to label endothelial and hematopoietic cells (Lineage neg (lin neg ). Lin neg cells were excluded by FACS with streptavidin-conjugated allophycocyanin (catalogue no. 554067; BD). Staining with anti-CD49f (catalogue no. 551129; BD) and CD24 (catalogue no. 553261; BD) was used to identify the mammary-stem/progenitor cell-enriched populations [34, (link)58] (link). Cell sorting was performed on a Beckman MoFlo Astrios EQ FACS sorter. To detect TAg expression FACS-sorted cells were cytospun onto slides, fixed and stained with anti-TAg (SV40 Large T Antigen (Cell Signaling (D1E9E) #15729).
Colony forming assays were performed as previously described [27] (link). Briefly, single-cell suspensions of EasySep-derived (STEMCELL Technologies) lin neg mammary epithelial cells were seeded in 20 l Matrigel (BD Biosciences) in Epicult-B medium (STEMCELL Technologies) supplemented with B27 (Gibco). Colony formation was scored after 12 days. The diameters of at least 30 of the largest acini were measured using Northern Eclipse software to calculate the mean and SEM.
Colony forming assays were performed as previously described [27] (link). Briefly, single-cell suspensions of EasySep-derived (STEMCELL Technologies) lin neg mammary epithelial cells were seeded in 20 l Matrigel (BD Biosciences) in Epicult-B medium (STEMCELL Technologies) supplemented with B27 (Gibco). Colony formation was scored after 12 days. The diameters of at least 30 of the largest acini were measured using Northern Eclipse software to calculate the mean and SEM.
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