G csf

Manufactured by Miltenyi Biotec

Sourced in United States, Japan, Germany, Sweden

G-CSF is a laboratory equipment product manufactured by Miltenyi Biotec. It is a recombinant form of the granulocyte colony-stimulating factor (G-CSF) protein, which is a key regulator of the production and function of neutrophils, a type of white blood cell. The core function of G-CSF is to stimulate the production and release of neutrophils from the bone marrow.

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Lab products found in correlation

12 protocols using g csf

Ex Vivo Expansion of Hematopoietic Stem Cells

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Sorted LT-HSCs, ST-HSCs or MEPs were cultured for 36–48 h in serum-free X-VIVO 10 (Lonza) media with 1% Bovine Serum Albumin Fraction V (Roche, 10735086001), 1× l-Glutamine (Thermo Fisher, 25030081), 1× Penicillin–Streptomycin (Thermo Fisher, 15140122) and the following cytokines (all from Miltenyi Biotec): FLT3L (100 ng/mL), G-CSF (10 ng/mL), SCF (100 ng/mL), TPO (15 ng/mL), and IL-6 (10 ng/mL). Cells were cultured in 96-well U-bottom plates (Corning, 351177).

Wagenblast E., Azkanaz M., Smith S.A., Shakib L., McLeod J.L., Krivdova G., Araújo J., Shultz L.D., Gan O.I., Dick J.E, & Lechman E.R. (2019). Functional profiling of single CRISPR/Cas9-edited human long-term hematopoietic stem cells. Nature Communications, 10, 4730.

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Monocyte Differentiation Protocol

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Monocytes were isolated from healthy donor leukopacs using a human CD14 isolation kit (Miltenyi) as per the manufacturer’s protocol. Monocytes were resuspended in RPMI complete without FBS and plated into a thermo-sensitive 6-well plate (Thermo Scientific 174901). 200 µl of human serum (either from healthy donors or patients) was added for a final concentration of 20% serum. 75 pg/mL of M-CSF (Peprotech) was added to each condition. Some monocytes were also cultured with 10 ng/mL of human IL-6 (Peprotech), 10 ng/mL GM-CSF (Peprotech), G-CSF (Miltenyi), IL-20 (Peprotech), or IL-33 (Peprotech). On days 3–5, the medium was changed, 400 µl of fresh RPMI complete without FBS was added and replenishment of serum and/or recombinant cytokines as indicated. On day 7, the cells were harvested using cold PBS aided by a cell scraper.

Cardot-Ruffino V., Bollenrucher N., Delius L., Wang S.J., Brais L.K., Remland J., Keheler C.E., Sullivan K.M., Abrams T.A., Biller L.H., Enzinger P.C., McCleary N.J., Patel A.K., Rubinson D.A., Schlechter B., Slater S., Yurgelun M.B., Cleary J.M., Perez K., Dougan M., Ng K., Wolpin B.M., Singh H, & Dougan S.K. (2023). G-CSF rescue of FOLFIRINOX-induced neutropenia leads to systemic immune suppression in mice and humans. Journal for Immunotherapy of Cancer, 11(6), e006589.

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Lentiviral Transduction of Activated T Cells

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T cells were stimulated with Dynabeads Human T-Activator CD3/CD28 (Thermo Fisher Scientific, San Diego, CA) and were expanded in T cell media (47.5% Click’s media [Irving Scientific, Santa Ana, CA], 47.5% RPMI-1640, 5% human serum, 2 mM L-glutamine, 100 IU/mL penicillin, and 100 μg/mL streptomycin) supplemented with 100 IU/mL IL-2. Forty-eight hours following stimulation, T cells were transduced with a lentivirus vector encoding ARI2h.³¹ (link) Subsequently, T cells were split and cytokines were refreshed every 1 to 2 days for a further 7 days of culture, unless indicated otherwise. For experiments in which T cells were exposed to G-CSF in culture, 10 ng/mL recombinant G-CSF (Miltenyi Biotech) was added to T cells that were resting in T cell media for 3 days prior to stimulation. ARP-1, U266, ARP-1-GFP-ffLuc, and U266-GFP-ffLuc cell lines were obtained, cultured, and modified to express GFP-ffLuc, where appropriate, as previously described.³¹ (link) All cultured cells were incubated at 37°C with 5% CO₂. Live cells were routinely counted using Trypan blue exclusion.

Battram A.M., Oliver-Caldés A., Suárez-Lledó M., Lozano M., Bosch i Crespo M., Martínez-Cibrián N., Cid J., Moreno D.F., Rodríguez-Lobato L.G., Urbano-Ispizua A, & Fernández de Larrea C. (2022). T cells isolated from G-CSF-treated multiple myeloma patients are suitable for the generation of BCMA-directed CAR-T cells. Molecular Therapy. Methods & Clinical Development, 26, 207-223.

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Hematopoietic Progenitor Assay of Transduced CD34+ Cells

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Vector transduced CD34+ cells were plated in 1 ml of methylcellulose medium (HSC-CFU media) supplemented with FBS, Bovine Serum Albumin (BSA), and different growth factors (e.g. GM-CSF, G-CSF, SCF, IL-3, IL-6, and Epo (Miltenyi Biotec, USA) and was performed in duplicate. Hematopoietic colony forming units (CFU-E, BFU-E, CFU-G and CFU-M) were scored after 14 days of culture. eGFP (fluorescent) colonies were identified by fluorescence microscopy.

Dighe N., Khoury M., Mattar C., Chong M., Choolani M., Chen J., Antoniou M.N, & Chan J.K. (2014). Long-Term Reproducible Expression in Human Fetal Liver Hematopoietic Stem Cells with a UCOE-Based Lentiviral Vector. PLoS ONE, 9(8), e104805.

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Isolation and Culture of Bone Marrow-Derived Myeloid-Derived Suppressor Cells

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Fresh BM aspirates were treated with K₂EDTA to prevent coagulation, and lysed to remove red blood cells with a hypotonic solution of ammonium chloride. Myeloid populations were isolated through magnetic sorting by the depletion of CD3⁺/CD19⁺/CD56⁺ lymphocytes, with a cocktail of immunomagnetic beads obtained by combining anti–human CD3, CD19, and CD56 beads (Miltenyi Biotec). Cell purity was checked by FACS analysis on forward/side scatter parameters. Subsequently, the CD3⁻/CD19⁻/CD56⁻ fraction was cultured with 40 ng/ml G-CSF and GM-CSF (Miltenyi Biotec) for 4 days at 37 °C, 8% CO₂ in order to obtain BM-MDSCs, as previously described [19 (link), 25 (link)]. On the fourth day, cells were collected and separated into CD11b⁻ and CD11b⁺ fractions with immunomagnetic anti–human CD11b beads (Miltenyi Biotec). The purity of sorted cells was checked by staining both fractions with anti-CD16 (BD Pharmingen) and anti-CD11b (Beckman Coulter) antibodies and analyzing cells by FACS Calibur cytometer (BD Biosciences). All the fractions were obtained with a purity of ≥90%.

Magri S., Masetto E., Solito S., Francescato S., Belluzzi E., Pozzuoli A., Berizzi A., Ruggieri P, & Mandruzzato S. (2020). Human MDSCs derived from the bone marrow maintain their functional ability but have a reduced frequency of induction in the elderly compared to pediatric donors. Immunity & Ageing : I & A, 17, 27.

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Hematopoietic Stem Cell Lineage Assay

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SLAM-HSC (150–200 cells), MPP (700–1500 cells), CMP/MEP (1000 cells), GMP (300–1000), PreMegE (1000 cells), and late MP (5000 cells) were plated in duplicate or triplicate in methylcellulose MethoCult 32/34 (Stemcell Technologies) with 10 ng/ml TPO (a generous gift from Kirin, Tokyo, Japan), 1 U/ml EPO (PreproTech), 10 ng/ml IL-3 (Miltenyi Biotec), 10 ng/ml IL-6 (Miltenyi Biotec), 100 ng/ml SCF (PreproTech), and 20 ng/ml G-CSF (Miltenyi Biotec). Colonies derived from erythroid progenitors (colony forming unit-erythroid [CFU-E]) were counted after 2 days, but no CFU-E was detected in any of the cell populations tested. Colonies derived from erythroid progenitors (burst forming unit-erythroid [BFU-E]), granulo-monocytic (colony forming unit-granulocyte macrophage [CFU-GM]), and multilineage colonies (mixed) progenitors were counted after 9 days. For megakaryocytic progenitor (CFU-MK) assay, SLAM-HSC (150–200 cells), MPP (2000 cells), CMP/MEP (2000 cells), GMP (2000), PreMegE (2000 cells), and late MP (5000 cells) were plated in triplicate in serum-free fibrin clot assays with SCF, IL-6, and TPO. MKs and CFU-MKs were evaluated at day 7 by acetylcholinesterase staining.

Tak T., Prevedello G., Simon G., Paillon N., Benlabiod C., Marty C., Plo I., Duffy K.R, & Perié L. (2021). HSPCs display within-family homogeneity in differentiation and proliferation despite population heterogeneity. eLife, 10, e60624.

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Cytogenetic Analysis of CD34+ Hematopoietic Cells

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Lineage depleted cord blood cells were thawed and CD34⁺ CD38⁻ cells were sorted using CD34 APC-Cy7 (clone 581, custom conjugation) and CD38 PE-Cy7 (335825, HB7). Cells were pre-cultured for 36–48 h in serum-free media and subsequently electroporated as described above. After overnight incubation, media was changed to IMDM (Thermo Fisher, 12440061) with 10% FBS (Sigma, 15A085), 1× l-Glutamine (Thermo Fisher, 25030081), 1× Penicillin–Streptomycin (Thermo Fisher, 15140122) and the following cytokines (all from Miltenyi Biotec): FLT3L (100 ng/mL), G-CSF (10 ng/mL), SCF (100 ng/mL), TPO (15 ng/mL), and IL-6 (10 ng/mL) to allow for cell expansion. Subsequently, karyotyping of chromosomes was performed according to standard procedures. Metaphase slides were prepared, then banded with trypsin and stained with Leishman’s stain. The G-banded slides were scanned and metaphases captured using an automated imaging system (MetaSystems). Metaphases were analyzed using Ikaros image analysis software (MetaSystems). For each condition, 20 metaphases were analyzed by G-banded karyotyping for numerical and structural abnormalities.

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Evaluating CD34+ Progenitor Cell Function

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In this experiment 1x10⁵ CD34⁺ cells were co-cultured in a volume of 500μl RPMI per well with or without MVs from 6 HD and 6 patients. After 24hours, 5x10³ cells were seeded into methylcellulose MACS Media with Stem cell Factor, GM-CSF, G-CSF, IL-3 and IL-6 (Miltenyi Biotec GmbH, Germany) to quantify the progenitor cell CFU-GM, as previously described[15 (link)].
These cultures were incubated in a humidified atmosphere at 37°C with 5% CO₂. After 14 days, CFU-GM colonies were scored with an inverted microscope. Results were expressed as the ratio between CFU-GM obtained with CD34⁺ cells that had been co-cultured with MVs from MDS or HD and the same CD34⁺ cells without MVs.

Muntión S., Ramos T.L., Diez-Campelo M., Rosón B., Sánchez-Abarca L.I., Misiewicz-Krzeminska I., Preciado S., Sarasquete M.E., de las Rivas J., González M., Sánchez-Guijo F, & del Cañizo M.C. (2016). Microvesicles from Mesenchymal Stromal Cells Are Involved in HPC-Microenvironment Crosstalk in Myelodysplastic Patients. PLoS ONE, 11(2), e0146722.

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Isolation and Culture of BM-MDSCs

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Fresh BM aspirates were treated with K 2 EDTA to prevent coagulation, and lysed to remove red blood cells with a hypotonic solution of ammonium chloride. Myeloid populations were isolated through magnetic sorting by the depletion of CD3 + /CD19 + /CD56 + lymphocytes, with a cocktail of immunomagnetic beads obtained by combining anti-human CD3, CD19, and CD56 beads (Miltenyi Biotec). Cell purity was checked by FACS analysis on forward/side scatter parameters. Subsequently, the CD3 -/CD19 -/CD56 -fraction was cultured with 40 ng/ml G-CSF and GM-CSF (Miltenyi Biotec) for 4 days at 37°C, 8% CO 2 in order to obtain BM-MDSCs, as previously described 19, 25 . On the fourth day, cells were collected and separated into CD11b -and CD11b + fractions with immunomagnetic anti-human CD11b beads (Miltenyi Biotec). The purity of sorted cells was checked by staining both fractions with anti-CD16 (BD Pharmingen) and anti-CD11b (Beckman Coulter) antibodies and analyzing cells by FACS Calibur cytometer (BD Biosciences). All the fractions were obtained with a purity of ≥ 90%.

Magri S., Masetto E., Solito S., Francescato S., Belluzzi E., Pozzuoli A., Berizzi A., Ruggieri P., & Mandruzzato S. (2020). Human myeloid-derived suppressor cells derived from the bone marrow maintain their functional ability with aging but have a reduced frequency of induction.

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Isolation and Characterization of Hematopoietic Progenitors

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Peripheral blood from patients was collected in citrate or EDTA tubes. No difference was observed between the two types of anticoagulant except that citrate allows a better platelet recovery. Granulocytes were obtained after hematopoietic cell separation on a ficoll gradient density. Hematopoietic progenitors (CD34 + ) were isolated from mononuclear cells (MNCs) by immunomagnetic enrichment (Miltenyi, Biotech). T cells (CD56 -CD14 -CD3 + ), B cells (CD56 -CD14 -CD19 + ), monocytes (CD14 + ), and NK cells (CD14 -CD56 + ) were sorted by the Influx flow cytometer (Beckton Dickinson) with a purity > 95%. The following cell fractions CD34 + CD38 -CD90 + , CD34 + CD38 -CD90 - and CD34 + CD38 + CD90 -were cloned at 1 cell/well and cultured in presence of a cocktail of human recombinant cytokines containing EPO (1 U/mL) (Amgen Thousand Oaks, CA), TPO (20 ng/mL) (Kirin, Japan), SCF (25 ng/ mL) (Biovitrum AB, Sweden), IL-3 (10 ng/mL), FLT3-L (10 ng/mL), G-CSF (20 ng/mL), and IL-6 (10 ng/mL) (MiltenyiBiotec). Fourteen days later, individual colonies were plucked and lysed with proteinase K and 0.2% Tween 20 (Sigma) at 65°for 60 min and 95 °C for 15 min. DNAs colonies were genotyped with two techniques, a conventional PCR amplification and a quantitative PCR amplification using specific primers for type 1 and type 2 as previously described (Table S2) [53] .

El-Khoury M., Cabagnols X., Mosca M., Vertenoeil G., Marzac C., Favale F., Bluteau O., Lorre F., Tisserand A., Rabadan Moraes G., Ugo V., Ianotto J.C., Rey J., Solary E., Roy L., Rameau P., Debili N., Pasquier F., Casadevall N., Marty C., Constantinescu S.N., Raslova H., Vainchenker W, & Plo I. (2020). Different impact of calreticulin mutations on human hematopoiesis in myeloproliferative neoplasms. Oncogene, 39(31).

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