OP9-DL1/OP9-GFP co-culture assays were essentially performed as described previously (48 (link)). Sorted precursors (DN2a and DN2b) were plated onto subconfluent OP9 BM stromal cells expressing either the Notch ligand Delta-like ligand 1 (OP9-DL1) or GFP (OP9-GFP) as a control in a 24-well plate. Cocultures were performed in the presence of 10 ng/mL SCF, 5 ng/mL Flt3-L, and 1 ng/mL IL-7 for OP9-DL1 T cell differentiation and 5 ng/mL IL-7 for OP9-GFP cultures (all obtained from Peprotech). After 4 days, half of the culture medium was replaced with fresh medium and at day 7 of differentiation thymocytes were harvested and separated from contaminating OP9 cells by filtering the cocultured cells through a 50 μm filter (Sysmex) prior to seed onto fresh OP9 monolayers. Lineage differentiation was monitored for up to 15 days. Discrimination of dead cells were performed using 7-amino-actinomycin D according to the manufacturer's instructions.
50 μm filter
Manufactured by Sysmex
The 50 μm filter is a laboratory equipment designed to separate and remove particles or substances from a liquid or gas sample. It features a pore size of 50 micrometers, which allows the passage of smaller components while retaining larger particles. The filter can be used in various applications that require sample preparation or purification.
Automatically generated - may contain errors
Lab products found in correlation
Moflo astrio, by Beckman Coulter (2 mentions)
Facsaria fusion, by BD (2 mentions)
Interleukin 7 (il 7), by Thermo Fisher Scientific (1 mentions)
α mem, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Gm csf, by R&D Systems (1 mentions)
U bottomed plate, by Corning (1 mentions)
Stem cell factor (scf), by Immunotools (1 mentions)
4 protocols using 50 μm filter
1
OP9 Co-Culture for T Cell Differentiation
2
Nuclei Isolation from Frozen Brain Tissue
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All human tissue protocols were approved by the Office for Human Research Protection at Sanford Burnham Prebys Medical Discovery Institute and conform to National Institutes of Health guidelines. Nuclei were prepared using nuclear extraction buffer (NEB) as described previously4 (link). Briefly, fresh frozen post-mortem brain tissue was sectioned at 50 μm using a cryostat and placed in 1 ml of ice-cold NEB for 10 minutes. Nuclei were extracted using a glass dounce homogenizer with Teflon pestle using 10–12 up-and-down strokes in 1 ml of NEB. Samples were passed through a 50 μm filter (Sysmex Partec), incubated on ice for 10 minutes. Samples were spun for 5 minutes at 250–300 × g, washed in PBS + 2 mM EGTA (PBSE), and resuspended in PBSE supplemented with 1% fatty-acid free bovine serum albumin (FAF-BSA, Gemini) containing 4′,6-diamidino-2-phenylindole (DAPI). DAPI+ single nuclei were purified by flow cytometry using MoFlo Astrios (Beckman Coulter) or FACSAria Fusion (Becton Dickinson), concentrated at 900 × g for 10 minutes and used directly for droplet encapsulation.
3
Nuclei Isolation from Frozen Brain Tissue
4
Differentiation of human CD34+ progenitors
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FACS-purified human PB or BM CD34+ progenitors, progenitor subsets or pre-DC were cultured in 96 well U-bottomed plates (Corning) with pre-seeded OP9 stromal cells (5000vwell) in 200 μL alpha-MEM (αMEM, GIBCO) supplemented with 1% penicillin/streptomycin (Sigma), 10% FCS, 20ng/ml granulocyte-macrophage colony-stimulating factor (GM-CSF, R&D systems), 100ng/ml Flt3-ligand (FLT3, Immunotools), 20ng/ml stem cell factor (SCF, Immunotools). CD34+ cells were seeded at 3000/well or 500/well for serial time points. Pre-DC were seeded at 500-3000 cells/well, determined by the number of cells available after FACS-purification. Half the volume of media, with cytokines, was replaced weekly. Cells were harvested on ice at day 14 or 21; or at days 3, 5, 7, 9, 11, 14 and 21 for serial time points, passed through a 50 μm filter (Sysmex Partec), washed in PBS, and stained for flow cytometric analysis or FACS-purification.
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