BM was harvested from the femur, tibia, and pelvis. Bones were fragmented by mortar and pestle in MACS buffer, and debris was removed by passing cells through a 70-µm strainer. Red blood cells were subsequently lysed with ammonium chloride-potassium bicarbonate lysis buffer. Cells were counted on a Vi-CELL analyzer (Beckman Coulter), and 3–5 × 106 cells were stained for analysis. For Flt3L culture experiments, whole BM (8 × 106 cells in 4 ml cIMDM) was cultured at 37°C with 100 ng/ml Flt3L (Peprotech) or equivalent volume PBS (vehicle) for 9 d. For comparative cytokine culture experiments, whole BM (6 × 106 cells in 4 ml of cIMDM) was cultured at 37°C with 100 ng/ml Flt3L (Peprotech), 20 ng/ml M-CSF (Peprotech), 50 ng/ml SCF (Peprotech), or equivalent volume PBS (vehicle) for 7 d. For all culture experiments, loosely adherent and suspension cells were harvested by gentle pipetting at the indicated time point and stained with fluorescent antibodies for analysis by flow cytometry.
Flt3l
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, Canada, Germany, Israel, Macao, Switzerland
Flt3L is a recombinant human protein that functions as a growth factor for hematopoietic progenitor cells. It is used in cell culture applications to support the proliferation and differentiation of various blood cell types.
Automatically generated - may contain errors
Lab products found in correlation
Stem cell factor (scf), by Thermo Fisher Scientific (134 mentions)
Interleukin 3 (il 3), by Thermo Fisher Scientific (93 mentions)
Interleukin 6 (il 6), by Thermo Fisher Scientific (73 mentions)
Interleukin 7 (il 7), by Thermo Fisher Scientific (62 mentions)
Gm csf, by Thermo Fisher Scientific (39 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (29 mentions)
G csf, by Thermo Fisher Scientific (27 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (24 mentions)
α mem, by Thermo Fisher Scientific (14 mentions)
Il 15, by Thermo Fisher Scientific (12 mentions)
Penicillin, by Thermo Fisher Scientific (12 mentions)
L glutamine, by Thermo Fisher Scientific (12 mentions)
Streptomycin, by Thermo Fisher Scientific (11 mentions)
Glutamax, by Thermo Fisher Scientific (11 mentions)
Stemspan sfem, by STEMCELL (10 mentions)
Il 11, by Thermo Fisher Scientific (10 mentions)
Thrombopoietin, by Thermo Fisher Scientific (10 mentions)
Stemspan, by STEMCELL (9 mentions)
M csf, by Thermo Fisher Scientific (8 mentions)
Retronectin, by Takara Bio (8 mentions)
316 protocols using flt3l
1
Bone Marrow Cell Isolation and Culture
2
Stimulation of NK cell cytokine production
3
Evaluating Hematopoietic Stem Cell Potential
4
Eosinophil Generation from Infected Mice
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Eosinophils were generated from cultures of bone marrow cells from infected and non-infected mice based on a previously described protocol (20 (link)). Briefly, bone marrow from the tibia and femur of BALB/c infected mice was used at 18 d.p.i. Cells were cultured at 0.5 × 106/mL in complete RPMI medium supplemented with 100 ng/mL SCF and 100 ng/mL FLT3-L (PeproTech). On day 4, the media containing SCF and FLT3-L was replaced with media containing 10 ng/mL IL-5 (PeproTech) alone and cultured for 10 days at 37°C and 5% CO2.
5
Flavonoid-Induced hiPSC Differentiation into NKCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The hiPSCs were differentiated into HPCs using the embryoid bodied (EB)-based HPC differentiation method [32 (link),33 (link)]. hiPSCs were briefly treated with the indicated flavonoids for 3 days before differentiation into EBs. For EB formation, cells were transferred onto Corning Ultra-Low Attachment Surface dishes with mTesR1 supplemented with 10 μM ROCK inhibitor for 6 days. On day 7, the formed EBs were transferred onto Matrigel-coated plate and incubated with the HPC differentiation medium (Iscove’s Modified Dulbecco′s Medium (Thermo Fisher Scientific) containing 20% FBS, with 100 ng/mL SCF (PeproTech), 10 ng/mL IL-3 (PeproTech), 10 ng/mL IL-6 (PeproTech), 20 ng/mL FLT3L (PeproTech), and 20 ng/mL BMP4 (PeproTech) with media exchange every 2 days until day 21. To obtain NKCs, hiPSC-derived HPCs were differentiated for 4 weeks in the presence of 10 ng/mL IL-15 (PeproTech), 5 ng/mL IL-3, 20 ng/mL IL-7 (PeproTech), 20 ng/mL SCF, and 10 ng/mL FLT3L. Medium containing cytokines was changed weekly with the exception of IL-3, which was only included for the first week of differentiation.
6
Efficient Dendritic Cell Differentiation from HoxB8 MPP
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HoxB8 MPP were differentiated into DC using a two-step protocol modified from Felker et al., 2010 (link) and described in Lutz et al., 2022 (link); Xu et al., 2022 (link). In brief, 0.75 million cells/ml were grown in MPP growth medium with 50 ng/ml Flt3L (Peprotech, 300–19) and reduced E2 (0.01 μM) for two days and cell density was kept to 0.75 million cells/ml. To induce DC differentiation, HoxB8 MPP were then washed with PBS to remove cytokines and E2, and cultured in RPMI 1640 medium supplemented with FCS, penicillin/streptomycin, L-glutamine, β-ME (same concentrations as above), and Flt3L (50 ng/ml, Peprotech) (referred to as DC differentiation day 0). Partial medium changes were performed on differentiation day 3 and 6. Spontaneous DC differentiation of HoxB8 MPP was achieved simply by removing E2 from growth medium (SCF, Flt3L, IGF1 and hyper-IL6), and culturing the cells at 1.5 million cells/ml (Lutz et al., 2022 (link); Xu et al., 2022 (link)).
7
Generating Plasmacytoid Dendritic Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Bone marrow (BM) pDCs were generated from C5aR1+/+ and C5aR1−/− mice using a modified protocol previously described (26 (link), 27 (link)). Briefly, BM cells were harvested from mouse femurs and tibias. BM cells (2 × 106) from C5aR1+/+ and C5aR1−/− mice were seeded in 6-well plates and cultured with RPMI 1640 medium containing 10% fetal calf serum, streptomycin (100 μg/mL), penicillin (100 U/mL), 2 μM glutamine, 50 μM β-mercaptoethanol, and recombinant mouse FLT3L (200 ng/mL; eBioscience, San Diego, CA, USA). Half culture medium was replaced every 5 days. In the another experiment, BM cells from C5aR1+/+ mice were simulated with FLT3L alone or the combination of FLT3L and recombinant mouse C5a (42 nM; Peprotech, Rocky Hill, NJ, USA), and medium group was used as the negative control. At day 10, detached cells were collected and analyzed the percentage of pDCs by FCM.
8
Isolation of Bone Marrow Macrophages and Plasmacytoid Dendritic Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BM cells were collected from the tibiae, femora, and pelvises of WT, Slc29a3−/−, and Slc29a3−/−Tlr7−/− mice, and red blood cells were removed using BD Pharm Lyse lysing buffer. For the preparation of BM-Mphs, BM cells were plated at a density of 7 × 106 cells per well on a non-tissue culture polystyrene 94-mm petri dish (Greiner Bio-One) and cultured in 10 ml RPMI medium (Gibco Thermo Fisher Scientific) supplemented with 10% FBS, penicillin-streptomycin-glutamine (Gibco Thermo Fisher Scientific), 50 μM 2-ME, and 100 ng/ml recombinant murine macrophage colony-stimulating factor (M-CSF; PeproTech, Inc.) for 6 d. The attached cells on petri dishes were collected and used as BM-Mphs. For BM-pDCs, BM cells were plated at a density of 2.5 × 107 cells per 10-cm cell culture dish (Greiner Bio-One) and cultured in 10 ml RPMI 1640 medium (Gibco) supplemented with 10% FBS, penicillin-streptomycin-glutamine, 50 μM 2-ME, and 100 ng/ml recombinant murine FMS-like tyrosine kinase-3 ligand (Flt3L, PeproTech, Inc.) for 7 d. Flt3L-induced pDCs were stained with anti-CD11c/B220 mAbs, and CD11c+B220+ cells were sorted as BM-pDCs using a FACSAria flow cytometer (BD Biosciences).
9
Differentiation Protocols for NK and GMP Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For NK cell differentiation, cells were cultured in RPMI-1640 (Sigma) supplemented with 10% fetal bovine serum (FBS, Gibco), Penicillin/Streptomycin (Gibco), human IL-15 (10 ng/ml, Peprotech), Flt-3L (10 ng/ml, Peprotech) and SCF (10 ng/ml, Peprotech), at 37 °C and 5% CO2. For GMP differentiation, CB CD34+ cells were cultured in RPMI 1640 supplemented with 10% FBS, IL-3 (5 ng/mL, Peprotech), IL-7 (20 ng/mL, Peprotech), IL-15 (10 ng/mL), SCF (20 ng/mL), and Flt-3L (10 ng/mL)9 (link), at 37 °C and 5% CO2.
10
Murine Bone Marrow-Derived Eosinophil Isolation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Bone marrows from the femur and tibia of 6- to 8-week-old mice were flushed with PBS and processed into a single-cell suspension. After red blood cells were lysed with red blood cell lysis buffer, cells were resuspended in bone marrow medium [RPMI 1640 containing 20% FBS, 55 μM β-mercaptoethanol, 10 mM nonessential amino acids, 1 mM sodium pyruvate, penicillin (100 IU/ml), streptomycin (100 μg/ml), 2 mM glutamine, and 25 mM Hepes buffer] at a concentration of 1 × 106 cells/ml. Cells were stimulated with stem cell factor (SCF; 100 ng/ml) and fms-like tyrosine kinase 3 ligand (FLT3L; 100 ng/ml) (both from PeproTech, Rocky Hill, NJ) for 4 days. On day 4, the medium containing SCF and FLT3L was replaced with a medium containing only rmIL-5 (10 ng/ml; PeproTech, Rocky Hill, NJ). On day 8, cells were transferred to new flasks and maintained in fresh medium supplemented with rmIL-5. Half of the medium was replaced with fresh medium containing rmIL-5 every other day, and the cell concentration was adjusted each time to 1 × 106 cells/ml. On day 14, BMDEs were stimulated with mIFNγ (15 ng/ml) and mTNF (15 ng/ml) for 16 hours to activate cells. BMDE purity and viability were analyzed by flow cytometry.
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!