Microglia were differentiated as previously described. Briefly, hiPSCs expressing six inducible transcription factors (MAFB, CEBPα, IRF, PU1, CEBPβ, IRF5) were grown in Essential 8™ Basal Medium (Gibco/Thermo Fisher Scientific, Cat. No. A15169–01), 10 μM ROCK inhibitor, and 2 μg/ml Doxycycline on Matrigel and 10 cm Poly-D-Lysine coated plates at a density of 1.5 million cells per dish. After 2 days, cells were grown in a differentiation media consisting of Advanced DMEM/F12 (Gibco/Thermo Fisher Scientific, Cat. No. 35050–061), 1X GlutaMAX, 2ug/ml doxycycline, 100 ng/mL Human IL34 (Peprotech; Cat. No. 200–34) and 10 ng/mL Human GM-CSF (Peprotech; Cat. No. 300–03). Two days later, media was exchanged for iTF-Microglia media consisting of Advanced DMEM/F12, 1X GlutaMAX, 2 μg/mL doxycycline, 100 ng/mL Human IL-34, 10 ng/mL Human GM-CSF, 50 ng/mL Human M-CSF (Peprotech; Cat. No. 300–25) and 50 ng/mL Human TGFB1 (Peprotech; Cat. No. 100–21C). On day 8, cells were dissociated with TypLE express (Gibco/Thermo Fisher Scientific, Cat. No. 12605–028) and seeded into iAssembloids.
Human gm csf
Manufactured by Thermo Fisher Scientific
Sourced in United States
Human GM-CSF is a recombinant protein that functions as a growth factor. It stimulates the production and differentiation of granulocytes and monocytes from bone marrow precursor cells.
Automatically generated - may contain errors
Lab products found in correlation
Human il 4, by Thermo Fisher Scientific (17 mentions)
Interleukin 4 (il 4), by Thermo Fisher Scientific (11 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (7 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (6 mentions)
Cd14 microbead, by Miltenyi Biotec (5 mentions)
L glutamine, by Thermo Fisher Scientific (5 mentions)
Streptomycin, by Thermo Fisher Scientific (5 mentions)
Human m csf, by Thermo Fisher Scientific (4 mentions)
Murine gm csf, by Thermo Fisher Scientific (3 mentions)
2 mercaptoethanol, by Thermo Fisher Scientific (3 mentions)
Penicillin, by Thermo Fisher Scientific (3 mentions)
Human flt3l, by Thermo Fisher Scientific (2 mentions)
Human scf, by Thermo Fisher Scientific (2 mentions)
Lipopolysaccharides (lps), by Merck Group (2 mentions)
Ifn γ, by Thermo Fisher Scientific (2 mentions)
Gm csf, by Thermo Fisher Scientific (2 mentions)
Medium 1640 glutamax, by Thermo Fisher Scientific (2 mentions)
Dexamethasone (dex), by Merck Group (2 mentions)
Human g csf, by Thermo Fisher Scientific (2 mentions)
L glutamine, by Lonza (2 mentions)
59 protocols using human gm csf
1
Generation of Inducible Microglia-like Cells
2
Generation of Human PBMC-Derived Dendritic Cells
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To prepare human PBMC-derived DCs, human monocytes were enriched by plastic adherence of PBMCs in a 100 mm Petri dish at 37 °C and 5% CO2. After 2 h of incubation, the nonadherent cells were reserved to sort CD8+ T cells with a Naive CD8+ T Cell Isolation Kit (Miltenyi Biotec, Cat: 130–093-244) for subsequent experiments. The remaining adherent cells were cultured in medium containing 100 ng/mL human GM-CSF (PeproTech, Cat: AF-300–03-50UG) and 20 ng/mL human IL-4 (InvivoGen, Cat: rcyec-hil4). After 48 h, the medium was refreshed, and 10 ng/mL TNF-α (InvivoGen, Cat: rcyc-htnfa) was added. On day 3, the human DC suspension was harvested.
3
Differentiation and Expansion of iNKT Cells
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APCs were cultured in supplemented RPMI-1640 culture medium (Sigma). Sorted human T cells were cultured in Iscove’s Modified Dulbecco’s Medium (Sigma) containing recombinant IL-2 (1,000 U/mL) and 5% human serum. CD14+ monocytes were supplemented with human IL-4 (500 U/mL) and 50 ng/mL human GM-CSF (Peprotech) upon monocyte isolation and were fully differentiated on day 5. MoDCs pulsed with αGC were cocultured with the autologous CD14− fraction. From this, iNKT cells were sorted and expanded as previously described. BM cells were plated at 2 million cells per well in a 6-well plate. Medium was replenished with fresh GM-CSF (20 ng/mL) every 2 d for 5 to 7 d for differentiation into CD11c+ BMDCs.
4
Differentiation of CD14+ Monocytes into Dendritic Cells
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CD14+ monocytes were initially purified from PBMCs by negative immunomagnetic selection according to the manufacturer's instructions (EasySepTM Human Monocyte Enrichment kit, STEMCELL Technologies). Purified monocytes were washed twice and cultured in six-well plates at a density of 0.5 × 106 cells/mL in 1.2 mL of RPMI-1640 medium (Lonza), supplemented with 2% normal human serum type AB (NHS AB), 600 µg/mL l -glutamine (Sigma), and 20 µg/mL gentamicin (Gibco). Cultures were established in the presence of 100 ng/mL human GM-CSF (Peprotech) and 20 ng/mL IL-4 (Peprotech). Cells were cultured at 37 °C, 95% humidity, and 5% CO2 for 6 days. During the cultivation, cultures were supplemented with fresh medium supplemented with GM-CSF and IL-4. At day 6, cells were functionally evaluated.
5
Cell Culture Protocols for Immunological Research
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Jurkat T cells (E6.1 clone; no. 88042803; European Collection of Authenticated Cell Cultures [ECACC]), HeLa cells (no. 93021013; ECACC), human primary dermal fibroblasts (C-013-5C; Thermo Fisher Scientific), and HEK293-FRT TRex cells stably expressing WT or mutant Sec61α were cultured in RPMI GlutaMAX (Jurkat) or DMEM GlutaMAX (other cells) from Thermo Fisher Scientific, supplemented with 10% heat-inactivated FCS (Invitrogen), 100 U/ml penicillin, and 100 µg/ml streptomycin. Human primary T cells were isolated from blood donors by Ficoll density gradient centrifugation and CD4+ T cell purification by negative depletion (Miltenyi Biotec). Human primary macrophages were obtained from peripheral blood-derived monocytes, isolated by adhesion to tissue culture plasticware, and cultured with 10 ng/ml human GM-CSF (PeproTech) for 7–12 d. Mouse CD3+ primary T cells were isolated from spleens and LNs by negative selection using the Pan T cell isolation kit (Miltenyi Biotec) and then placed in RPMI medium supplemented with 10% heat-inactivated FCS, 10 mM Hepes, 1 mM pyruvate, and 25 µM 2-mercaptoethanol. Bone marrow–derived macrophages were obtained by a 7-d differentiation of mouse progenitors in DMEM supplemented with 20% heat-inactivated horse serum (Gibco) and 30% L929-conditioned medium as a source of M-CSF.
6
Differentiation of GFP⁺CD34⁺ HSCs into DCs
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Sorted GFP+CD34+ HSCs were allowed to expand for a further 72 h in X‐VIVO‐15/FCS/cytokine media at 2.5 × 104 mL−1 before differentiation into DCs according to a modified published protocol.30 In brief, the expanded cells were cultured at a density of 6.25 × 104 mL−1 in RPMI 1640, 10% FCS, 1 mm HEPES, 1 mm sodium pyruvate, 100 U mL−1 penicillin‐streptomycin, 2 mm Glutamax (all Thermo Fisher Scientific) and 50 µm β‐mercaptoethanol (Sigma‐Aldrich, St Louis, MO, USA) supplemented with 100 ng mL−1 human FLT3L, 20 ng mL−1 human SCF, 2.5 ng mL−1 human IL‐4, 2.5 ng mL−1 human GM‐CSF (all cytokines from Peprotech) and 1 µm StemRegenin 1 (STEMCELL Technologies, Vancouver). Media and cytokines were replenished on Day 6 of the differentiation culture and harvested for analysis by flow cytometry after 12 days.
7
Monocyte-Derived Dendritic Cell Activation
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Peripheral blood mononuclear cells (PBMC) from healthy donors (Sanquin) were isolated by centrifugation over a Ficoll gradient. Using magnetic CD14 Microbeads (Miltenyi), the CD14+ fraction was isolated from these PBMC. The CD14+ cells were cultured for 5 days in the presence of 800 U/ml human GM-CSF (Peprotech) and 500 U/ml human IL-4 (Peprotech). On day 5, the monocyte-derived DC (moDC; CD11c+ CD1a+ CD14− HLA-DRlo) were stimulated with the indicated reagents for 48 hours. The concentration of IL-12p40 in the supernatant after 24 hours was measured by ELISA (BioLegend). After 48 hours, the moDC were stained with fluorescently labeled antibodies targeted to CD83, CD86 and HLA-DR (eBioscience), and analyzed by flow cytometry (BD).
WT and TLR2-expressing HEK293 cells (Invivogen) were cultured in IMDM supplemented with 100 IU/ml penicillin/streptomycin (Gibco), 2 mM L-glutamin (Gibco) and 8% fetal calf serum (FCS; PAA Laboratories). TLR2-HEK293 cells were cultured in the presence of 10 μg/ml of the selective antibiotic blasticidin. In a 96 wells plate, 20,000 HEK293 or TLR2-HEK293 cells were seeded per well and stimulated with titrating doses of the indicated compounds in duplicates. After 24 hours of incubation at 37°C, supernatant was taken from all wells and the concentration of IL-8 was measured by ELISA (BioLegend).
WT and TLR2-expressing HEK293 cells (Invivogen) were cultured in IMDM supplemented with 100 IU/ml penicillin/streptomycin (Gibco), 2 mM L-glutamin (Gibco) and 8% fetal calf serum (FCS; PAA Laboratories). TLR2-HEK293 cells were cultured in the presence of 10 μg/ml of the selective antibiotic blasticidin. In a 96 wells plate, 20,000 HEK293 or TLR2-HEK293 cells were seeded per well and stimulated with titrating doses of the indicated compounds in duplicates. After 24 hours of incubation at 37°C, supernatant was taken from all wells and the concentration of IL-8 was measured by ELISA (BioLegend).
8
Antibody-Mediated Macrophage Activation
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Example 3
In a third experiment, isolated human monocytes were cultured in complete RPMI medium (10% FBS, P/S, glutamine) supplemented with human GM-CSF (PeproTech, Inc.). Following 6 days of incubation, macrophages were harvested and added to TC-treated 96 well plates (Corning Inc., Corning, N.Y.), wherein the macrophages were stimulated with a dose titration of each of the indicated antibodies. After an 18 hour incubation, supernatants were collected and TNFα levels were measured by ELISA according to the manufacturer's instructions (Invitrogen eBioscience Human TNF alpha ELISA Ready-SET-Go!). As can be seen in
Specifically, R3H-P1F05 comprises SEQ ID NOs: 326 and 334, R3H-P1F05 hIgG1-G236A comprises SEQ ID NOs 326 and 335, R3H-P1F05 hIgG1-LALA comprises SEQ ID NOs: 326 and 336, R3H-P1F05 hIgG1-N297A comprises SEQ ID NOs: 326 and 337, and R3H-P1F05 hIgG1-C219S comprises SEQ ID NOs: 326 and 338.
9
Isolation and Culture of Monocytes
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Blood was collected during exsanguination. Some of the peripheral blood leukocytes were subjected to flow cytometry in order to evaluate the frequency of CD3, CD19, CD33, CD45, and BDCA-3 positive cells. Remaining blood, spleen, and bone marrow were used for an independent separation of intact monocytes using the negative section with the use of magnetic beads as described before (17 (link), 27 (link)). The purity of the cells exceeded 95% for CD33(bright) and 80% for CD14(+). Isolated MO were incubated in X-VIVO 10/15™ media with gentamycin and phenol red (Lonza, Cohasset, MN, USA), supplemented with human IL-4 (Peprotech, Rocky Hill, NJ, USA) at 500 IU/ml and human GM-CSF (Peprotech, Rocky Hill, NJ, USA) at 1,000 IU/ml at 37°C and 5% CO2. On day 3, 50% of the supernatant was collected and replenished with fresh media and cytokines. After 5 days, the supernatants and cells were collected using an ice-cold 10 mM EDTA buffer (Gibco, Grand Island, NY, USA), followed by a washout with PBS without Ca2+ or Mg2+ (Gibco, Grand Island, NY, USA) (27 (link)). In some experiments, rabbit polyclonal neutralizing antibody for M-CSF (FN M-CSF, 5 µg/ml) was added at the beginning of the culture. The dose was shown to fully neutralized M-CSF in prior study (17 (link)). An addition of non-specific IgG had no effect on MO function (17 (link)).
10
Dendritic Cell Generation From Mouse Bone Marrow
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Mouse bone marrow (BM) -derived DCs were generated as previously reported48 (link). Briefly, BM progenitors isolated from the femurs and tibias were incubated at 5 × 105 cells/ml in generating medium (RPMI 1640 containing 10% FBS [Hyclone], 2 mmol/L glutamine, 25 mmol/L HEPES, 100 U/ml penicillin, 100 μg/ml streptomycin, and 50 μmol/L 2-mercaptoethanol and 20 ng/ml of mouse GM-CSF (PeproTech) at 37 °C in a humidified incubator with 5% CO2 for 6 d to generate immature DCs. Subsequently, immature DCs were incubated in fresh culture medium at 5 × 105 cells/ml in the absence (sham) or presence of HMGN1, R848 (InvivoGen), or LPS (Sigma-Aldrich, as positive control) alone or in combination at specified concentrations for 6–48 h at 37 °C in a humidified incubator with 5% CO2 before analyzing their function and phenotype. Recombinant HMGN1 used in the present study was generated in insect cells using a baculovirus expressing system and purified as previously described27 (link).
For the generation of human DCs, peripheral blood monocytes were isolated and cultured in the presence of 50 ng/ml of human GM-CSF (PeproTech) and IL-4 (PeproTech) for 5 to 7 days as previously described48 (link).
For the generation of human DCs, peripheral blood monocytes were isolated and cultured in the presence of 50 ng/ml of human GM-CSF (PeproTech) and IL-4 (PeproTech) for 5 to 7 days as previously described48 (link).
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