In total, 50 000 total FL cells were obtained from Ryk WT and Ryk KO mice and were co-cultured on confluent layers of OP9 WT/DL1, OP9 Wnt3a/DL1, or OP9 Wnt5a/DL1 mixed in a 1:1 ratio as described previously27 (link) with Alpha MEM 10% FCS containing 50 ng/ml rmSCF, 10 ng/ml rmFlt3L and 10 ng/ml rmIL-7 (all cytokines from R&D Systems, Abinsdon, UK) in 24-well plates. Cells were harvested after 7 and 14 days of co-culture and assessed for T-cell development by flow cytometric analysis. FTOC were done as described before47 (link) using fetal thymic lobes from E14 embryos, which were genotyped for the status of the Ryk deficiency. Thymic lobes were cultured on a nitrocellulose filter on air/medium interphase for 7–14 days, dispersed, and analyzed by flow cytometry.
Rmflt3l
Manufactured by R&D Systems
Sourced in United States
RmFlt3L is a recombinant mouse Fms-like tyrosine kinase 3 ligand (Flt3L) protein. Flt3L is a growth factor that stimulates the proliferation and differentiation of hematopoietic stem cells and progenitor cells.
Automatically generated - may contain errors
Lab products found in correlation
Rmscf, by R&D Systems (4 mentions)
Rmil 7, by R&D Systems (2 mentions)
Fetal calf serum (fcs), by Merck Group (1 mentions)
Magnetic activated cell sorting separation columns, by Miltenyi Biotec (1 mentions)
5 n ethylcarboxamidoadenosine neca, by Bio-Techne (1 mentions)
Accutase, by Merck Group (1 mentions)
Flow cytometry staining buffer, by Thermo Fisher Scientific (1 mentions)
Lipopolysaccharide, by InvivoGen (1 mentions)
Tlrl eblps, by InvivoGen (1 mentions)
Rmgm csf, by R&D Systems (1 mentions)
Retronectin, by Takara Bio (1 mentions)
Stemspan serum free expansion medium, by STEMCELL (1 mentions)
Rmtpo, by R&D Systems (1 mentions)
Rmscf, by Thermo Fisher Scientific (1 mentions)
Torin1, by Bio-Techne (1 mentions)
Sodium pyruvate, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Non essential amino acids (neaa), by Merck Group (1 mentions)
L glutamine, by Merck Group (1 mentions)
β mercaptoethanol, by Merck Group (1 mentions)
6 protocols using rmflt3l
1
Ryk-mediated T-cell development in vitro
2
OP9 Co-Culture Conditions for Hematopoietic Differentiation
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For different experiments various conditions of OP9 co-cultures were used as follows: OP9-WT, OP9-DL1-GFP, OP9-WT/DL1 (1 : 1), OP9-DLW3A, OP9-DLW5A, OP9-DLW3A/DL1 (1:1), OP9-DLW3A/DL1 (1 : 10) and OP9-DLW3A/DL1 (1 : 100). In all conditions 50 000 total fetal liver cells were cultured on confluent layers of OP9 cells, with AlphaMEM 10% FCS containing 50 ng/ml rmSCF, 10 ng/ml rmFlt3L and 10 ng/ml rmIL-7 (all cytokines from R&D systems, Minneapolis, MN, USA) in a well of 24-wells plate. For different purposes, cells were harvested after 6 h, 24 h, 3, 7 or 14 days of culture and stained for flow cytometric analysis.
3
Isolation and Purification of Siglec-H KO Mouse pDCs
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BM cells were isolated from femur and tibia. After red blood cell lysis with red blood cell lysis solution, BM cells from WT and Siglec-H KO mice were cultivated in complete RPMI (50 µM 2-mercaptoethanol, 1% nonessential amino acids, 1 mM sodium pyruvate, 100 µg/ml kanamycin sulfate, and 10% FCS [Sigma-Aldrich]) with 100 ng/ml rmFlt3L (R&D Systems) for 8 d at 2 × 106 cells/ml (25-cm2 cell culture flasks, each with 5 ml of cells). After 4 d, 2.5 ml of medium per flask was replaced by 2.5 ml of fresh medium containing 50 ng/ml rmFlt3L. After 8 d, CD11c+PDCA+ pDCs were purified with magnetic-activated cell-sorting separation columns (Miltenyi Biotec).
4
Generation of Murine Dendritic Cell Subsets
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The generation of CD11c+ CD103+ CLEC9A+ DC was described previously.36 (link) Briefly, bone marrow was aseptically isolated from femurs and tibiae of untreated C57BL/6 mice and cultured in complete T cell medium for 16 days with the addition of 200 ng/mL rmFLT3L and 5 ng/mL rmGM-CSF (R&D Systems cat# 427-FL-005 and cat# 415 ML, respectively). Where indicated, after 10 days of initial culture with growth cytokines, AZD4635 at 3 µM, or DMSO as control was added and allowed to incubate for 30 min prior to addition of 5 µM 5′-(N-ethylcarboxamido) adenosine (NECA) (Tocris). The cultures were then continued with growth cytokines for the remaining 6 days prior to cell harvest. Immature dendritic cells (iDC) were induced to a mature phenotype with 1 µg/mL lipopolysaccharide (InvivoGen cat# tlrl-eblps) for 24 hours. Both iDC and mature DC were used for experiments where indicated. Before use in subsequent assays such as T cell co-cultures, DC were washed with Dulbecco’s PBS (DPBS), harvested with Accutase (Sigma cat# A6964) and then washed and resuspended in complete T cell media or flow cytometry staining buffer (ThermoFisher Scientific cat# 00-4222-26).
5
Retroviral Transduction of Hematopoietic Progenitors
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LSK and LK sorted cells were stimulated overnight in StemSpan serum-free expansion medium (STEMCELL Technologies) supplemented with rmTPO (recombinant mouse thrombopoietin) (10 ng/ml; R&D Systems), rmFlt3L (50 ng/ml; R&D Systems), and rmSCF (100 ng/ml; R&D Systems). Hematopoietic progenitors were transduced using RetroNectin (Takara Bio Inc.)–coated wells according to the manufacturer’s instructions. Nontissue culture plates were coated with RetroNectin overnight at 4°C and then blocked with 2% BSA in PBS for 30 min. Retroviral supernatant (24 or 48 hours) was centrifuged at 1500g for 1 hour at 32°C and incubated an extra hour at 37°C. After coating, viral supernatant was removed and stimulated cells were immediately added on the virus-coated plates. Cells were cultured in StemSpan medium supplemented with rmTPO (10 ng/ml), rmFlt3L (50 ng/ml), and rmSCF (100 ng/ml) and transduced overnight at 37°C. LZRS-ires-eGFP–, LZRS-Gata3-ires-eGFP–, and LZRS-Bcl11b-ires-eGFP–transduced cells were used for in vitro and in vivo approaches.
6
Isolation and Culture of Mouse Eosinophils
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The protocols, isolation, and culture of mouse eosinophils were fully described elsewhere25 (link)–28 (link). Generally, bone-marrow derived non-adherent mononuclear cells (NAMNCs) were seeded at 1 × 106/ml in IMDM completed medium containing IMDM (Iscove’s modified Dulbecco’s medium; Invitrogen, Waltham, MA, USA) with 20% FBS (Gibco; origin from Australia), 100 IU/ml penicillin and 10 mg/ml streptomycin, 2mM L-glutamine, 1 × non-essential amino acids (Sigma-Aldrich, St. Louis, MO, USA), 1 mM sodium pyruvate (Sigma-Aldrich) and 0.006‰ β-mercaptoethanol (Sigma-Aldrich). 100 ng/ml rmFlt-3L (Peprotech, Rocky Hill, NJ, USA), and 100 ng/ml rmSCF (Peprotech) were supplemented from day 0 to day 4. Medium was replaced on day 4 and day 8, containing 10 ng/ml rmIL-5 (R&D Systems, Minneapolis, MN, USA), but without rmFlt-3L and rmSCF. Most experiments were performed in day 8 to day 10. Torin-1 (Tocris Biosciences) and U0126 (Selleck) were treated from day 4 when the medium was replaced. Cells were harvested and detected using PE-conjugated anti-SiglecF, and apoptotic levels were analyzed using AnnexinV-FITC and 7-AAD. Cells were lysed and detected by western blotting with p-S6 (Cell signaling technology, Denver, MA, USA), LC3B, Erk1/2, p-Erk1/2 and β-actin and analyzed Mbp and Gata-1 mRNA levels using quantitative real-time PCR.
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