Gibco™ RPMI 1640 Medium

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Use of decellularized thymus scaffolds

Protocol tips
Use of decellularized thymus scaffolds

Publication protocol

Thymus organoid reconstruction. To reconstruct the thymus organoids, thymic tissue was harvested from 2- to 3-week-old B6 mice, unless specified otherwise, and separated into single cells with collagenase digestion as described earlier.32,67 In brief, thymic tissue (n = 3–4) was pooled and needle dissected into small pieces of ~1 mm3, and digested with the Liberase TM solution [0.025 mg/ml Liberase TM (Roche Applied Science, Indianapolis, IN), 0.2 mg/ml DNase I (Roche Applied Science), and 10 mmol/l HEPES in RPMI-1640 (Life Technologies, Carlsbad, CA)] at 37oC for a total of 18 minutes (three rounds of 6 minutes each). All fractions were pooled, incubated with magnetic bead-conjugated anti-CD45 antibodies, and subjected to negative selection of CD45– TSCs (e.g., TECs and thymic fibroblasts) with MACS separation technology (Miltenyi Biotec, Auburn, CA). Approximately 0.25–0.5 million stromal cells were routinely obtained per thymus. Lin– progenitors were enriched from BM cells of 2–3-week-old B6.CD45.1 congenic mice (unless specified otherwise) with mouse Lineage cell depletion kit (Miltenyi Biotec), following the manufacturer's instructions. Lin– progenitors and TSCs were mixed at 1 : 1 ratio and resuspended in RPMI-10 solution at a concentration of 5 × 107/ml. A 10 μl of cell mix (~0.5 × 106 cells) was injected into the decellularized thymus scaffolds using syringes with 32-G needles or pulled glass needles under the dissection scope. The reconstructed thymus organoids were cultured in the top chamber of a 3.5-cm transwell in RPMI-10 medium before being transplanted underneath the kidney capsules of 10–16-week-old B6.nude mice. To evaluate T-cell development in the reconstructed thymus organoids, BM progenitors harvested from the B6 mice were mixed with TSCs isolated from C57BL/6.CD45.1 thymi at 1 : 1 ratio. The reconstructed thymus organoids were cultured in the upper chambers of 24-well transwell plates (one thymus per well; Corning, Tewksbury, MA) in complete RPMI-10 medium supplemented with 2 ng/ml interleukin-7 (Miltenyi Biotec) for 9 days. At the end of the culture, the thymus organoids were digested with Dispase I (0.6 U/ml, Roche Diagnostic, Indianapolis, IN) for 15 minutes at 37 °C. The isolated cells were stained with anti-CD45.2, anti-CD3, anti-CD4, and anti-CD8 antibodies for FCM analysis.

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing 3D Cell Culture Media Mouse thymic organoids using Gibco™ RPMI 1640 Medium from Fisher Scientific.

Manufacturer protocol

Download the product protocol from Fisher Scientific for Gibco™ RPMI 1640 Medium below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing 3D Cell Culture Media Mouse thymic organoids using Gibco™ RPMI 1640 Medium from Fisher Scientific. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.