All experiments involving mice were reviewed and approved by the Institutional Animal Care and Use Committee. Bone marrow from 4–6 week old female C57BL/6 mice was harvested, lineage depleted, and cultured in the presence of recombinant murine SCF (rmSCF, Peprotech, 50ng/ml), IL-3 (rmIL3, Peprotech, 50ng/ml), and IL-6 (rmIL6, Peprotech, 50ng/ml) for 24 hours prior to transduction on RetroNectin (Takara Bio Inc.) coated plates. Cultured supernatants containing ecotropic envelope pseudotyped retroviral vectors were produced by transient transfection of 293T cells as previously described33 (link). Murine bone marrow cells were harvested 48 hours following transduction, sorted for vector-encoded mCherry or GFP expression, and plated on methylcellulose containing IL-3, IL-6, SCF and erythropoietin (EPO) (Stem Cell technologies, Vancouver, BC) as per manufacturer’s instructions. Colonies were counted after 7 days of growth at 37°C, harvested and serially replated.
Rmscf
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
The RmSCF is a compact and self-contained lab equipment that provides precise temperature control for a wide range of laboratory applications. Its core function is to maintain consistent and stable temperature conditions within a designated space or sample holder.
Automatically generated - may contain errors
Lab products found in correlation
Rmil 3, by Thermo Fisher Scientific (12 mentions)
Rmflt3l, by Thermo Fisher Scientific (9 mentions)
Rmil 6, by Thermo Fisher Scientific (7 mentions)
Rmtpo, by Thermo Fisher Scientific (5 mentions)
Rhil 6, by Thermo Fisher Scientific (4 mentions)
Rmil 7, by Thermo Fisher Scientific (3 mentions)
Stemspan sfem medium, by STEMCELL (3 mentions)
Rmgm csf, by Thermo Fisher Scientific (3 mentions)
Rmil 5, by R&D Systems (2 mentions)
Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (2 mentions)
Plat e packaging cells, by Cell Biolabs (2 mentions)
M3134, by STEMCELL (2 mentions)
Puromycin, by Merck Group (2 mentions)
Bit 9500, by STEMCELL (2 mentions)
β mercaptoethanol, by Merck Group (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
B27 supplement, by Thermo Fisher Scientific (2 mentions)
Primovert, by Zeiss (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
L glutamine, by Merck Group (2 mentions)
29 protocols using rmscf
1
Retroviral Transduction of Murine Hematopoietic Cells
2
Eosinophil Generation and Macrophage Co-culture
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Eosinophils were generated from BMCs as described previously (Dyer et al., 2008 (link)). In brief, BMCs (1 × 106/ml) were cultured in Iscove's modified Dulbecco's medium containing 20% FBS, 100 IU/ml penicillin and 10 μg/ml streptomycin, 2 mM glutamine and 1x non‐essential amino acids and 1 mM sodium pyruvate and 50 μM β‐mercaptoethanol supplemented with 100 ng/ml recombinant murine stem‐cell factor (rmSCF, PeproTech, RockyHill, NJ, USA) and 100 ng/ml fms‐like tyrosine kinase 3 ligand (rmFLT3L, PeproTech) from day 0 to day 4. On day 4, the medium was replaced with a medium containing 10 ng/ml rmIL‐5 (R&D Systems, Minneapolis, MN, USA) only and was changed with 10 ng/ml rmIL‐5 every other day until day 12.
To carry out co‐culture of bone marrow macrophages with eosinophil‐CM, CM from day 7 to day 8 (24 h) of ex vivo eosinophil cultures derived from WT or mS6KO were collected. BMCs from C57BL/6 mice were incubated with M‐CSF for 48 h followed by 8 h‐incubation with CM, and mRNA of M2 macrophage genes was analyzed.
To carry out co‐culture of bone marrow macrophages with eosinophil‐CM, CM from day 7 to day 8 (24 h) of ex vivo eosinophil cultures derived from WT or mS6KO were collected. BMCs from C57BL/6 mice were incubated with M‐CSF for 48 h followed by 8 h‐incubation with CM, and mRNA of M2 macrophage genes was analyzed.
3
Modulation of Mast Cell Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BMMCs, PCMCs, or the p65/RelA-eGFP MC/9 reporter MCs were washed to remove IL-3 and were seeded in IL-3-free media at a density of 1 × 106 cells/mL. After 1 h, the cells were treated with vehicle (DMSO) or the HSP90 inhibitor 17AAG (the concentrations are indicated in the figures) for 30 min. Subsequently, the cells were stimulated with rmSCF and/or rmIL-33 (50 ng/mL) (Peprotech).
4
Flow Cytometry Purification of Thymocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Pre-leukemic and leukemic thymocytes were purified by flow cytometry and co-cultured on GFP-positive OP9 and OP9-DL1 stromal cell lines, as described previously91 (link). Thymocytes were co-cultured in reconstituted alpha-MEM medium (#12318, Gibco, Grand Island NY, USA) supplemented with 20% FCS (#12318), HEPES 10 mM (#15630-080), sodium pyruvate 1 mM (#11360-070), β-mercaptoethanol 55 μM (#21985-023), Glutamax 2 mM (#15750-060), 1% Penicillin/Streptomycin (#15140-122), 5 ng/mL rmFLT-3 ligand (#250-31 L, Pepro Tech), 5 ng/mL rmIL-7 (#217-17, Pepro Tech) and 25 ng/mL rmSCF (#250-03, Pepro Tech). After 48 h of co-culture, thymocytes were counted and analysed by FACS.
5
Retroviral Transduction of Mouse Hematopoietic Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell culture and retroviral transduction were performed as previously described with minor modification (Rathinam et al., 2010 (link)). Mouse hematopoietic cells were cutlred in RPMI (Thermo Fisher Scientific) with 10% FBS, 1% PS, 10ng/ml rm-IL3, rm-IL6, rm-TPO, rm-Flt3L and rm-SCF (PEPROTECH). For viral transduction, Cells were centrifused at 30 degree for 90 minutes at 2000 rpm with polybrene (8ug /ml). Plat-E packaging cells (Cell Bio labs) were used for retrovirus production. For making retrovirus plasmid PGFP-RV-mKlf1, mouse Klf1 cDNA was cloned from pMXs-ms-Klf1 (a gift from Shinya Yamanaka, Add gene plasmid #50785) (Nakagawa et al., 2008 (link)) into PGFP-RV.
6
Myeloid and Erythroid Differentiation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
De novo isolated or post–48-h liquid culture cells were plated in MethoCult GF M3434, supporting myeloid and erythroid differentiation, or MethoCult GF M3630, supporting preB cell differentiation (STEMCELL Technologies), supplemented with 0–20 ng/ml rmFlt-3L and 0–100 ng/ml rmSCF (PeproTech), and cultured at 37°C and 5% CO2. Colonies were scored between days 7 and 10 after plating, and images were captured using an inverted microscope (Diaphot 200; Nikon) at 4× with SPOT imaging software (v.5.0.15). Single representative colonies were isolated and used to confirm cell lineage by FACS analysis for the surface markers CD11b (M1/70), Gr-1 (RB6-8C5), and B220 (RA3-6B2) and Wright-Giemsa staining of fixed cytospins. Cytospin images were captured using an inverted microscope (Eclipse E200-LED; Nikon) at 100× with SPOT imaging software.
7
Erythroid Progenitor Assays from Mouse Bone Marrow and Human CD34+ Cells
8
Lentiviral Knockdown and Flow Cytometry of LSK Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
LSK cells were transduced overnight with control or gene-specific shRNAs and then cultured at 15,000 cells/well in non-tissue culture–treated 96-well plates for 5–6 d in serum-free expansion medium (STEMCELL Technologies) with 10 ng/ml RM SCF, 20 ng/ml RM Tpo, 20 ng/ml RM IGF-2 (PeproTech), 10 ng/ml RH FGF-1 (R&D Systems), and 10 µg/ml heparin (Sigma-Aldrich). Cells were collected 5–6 d after plating and stained with the following antibodies: (B220, CD3, CD4, CD8, CD19, Gr-1, and Ter119)-PerCP, Sca-1-PerCP-Cy5.5, and c-Kit-APC-780. After staining for surface antigens, cells were labeled with Annexin V-FITC (BD) and DAPI, and then analyzed using an LSR Fortessa and FlowJo version 9.4.11.
9
Single Cell Culture and Expansion
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Single cells were sorted and cultured in 96-well plates in StemSpan SFEM medium (STEMCELL Technologies, Cat. 09600) supplemented with 20 ng/ml rmSCF (Peprotech, 250-03), 20 ng/ml rmTPO (eBioscience, 34-8686-63), 20 ng/ml rmIl3 (Peprotech, 213-13), and 5 U/ml rhEpo (Roche) and cultivated for 12 days at 37 °C with 5% CO2.
10
Murine hematopoietic stem/progenitor cell culture
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
M-ATOs were generated as previously described (27 (link)). MS5-mDLL4 cells were harvested by trypsinization and resuspended in serum free M-ATO culture medium (“D/F12-B27”) composed of DMEM-F12 (Gibco, Cat# 11320033), 2% B27 supplement (ThermoFisher Scientific, Grand Island, NY, Cat# 17504-044), 30 µM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Sigma-Aldrich, St. Louis, MO, Cat# A8960-5G) reconstituted in 1X PBS, 1% penicillin/streptomycin (Gemini Bio-Products, West Sacramento, CA, Cat# 400-109), 1% Glutamax (ThermoFisher Scientific, Grand Island, NY, Cat# 35050-061), 5 ng/ml rmFLT3L (Peprotech, Rocky Hill, NJ, Cat# 250-31L), 5 ng/ml rmIL-7 (Peprotech, Cat# 217-17), 10 ng/ml rmSCF (Peprotech, Cat# 250-03) (of note SCF was added only for the first week of culture) and beta mercaptoethanol (bME) (0.05mM) (Sigma-Aldrich, Cat# M7522). D/F12-B27 was made fresh weekly. 1.5x105 MS5-mDLL4 cells were combined with purified murine HSPCs (100-4000 cells/ATO). M-ATOs were plated on a 0.4 µm Millicell transwell insert (EMD Millipore, Billerica, MA; Cat. PICM0RG50) placed in a 6-well plate containing 1 ml D/F12-B27 per well. Medium was changed completely every 3-4 days by aspiration from around the cell insert followed by replacement with 1 ml with fresh D/F12 and cytokines.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!