Penicillin streptomycin, by Miltenyi Biotec - Product details

1

Isolation and Expansion of Human CD34+ Cells

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Mononuclear cells were isolated from umbilical cord blood from anonymous healthy donors by density centrifugation using Ficoll (Biocoll, Merck Millipore). Human CD34+ cells were then enriched in the sample by immunomagnetic beads using an AutoMACSpro (Miltenyi Biotec). After collection, enriched CD34+ cells were frozen in a cryopreservation medium containing 90% of fetal bovine serum (Eurobio) and 10% of dimethylsulfoxide (Sigma) and stored in liquid nitrogen.
After thawing, the CD34+ cells were cultured in a 96-well plate in a humidified 5% CO2 incubator at 37°C. Cells were cultured in prestimulation medium made of XVivo (Lonza) supplemented with penicillin/streptomycin (respectively, 100 U/mL and 100 μg/mL; Gibco, Thermo Fisher Scientific), 50 ng/ml h-FLT3-ligand, 25 ng/ml h-SCF, 25 ng/ml h-TPO, and 10 ng/ml h-IL3 (Miltenyi) final concentration.

Parmentier R., Racine L., Moussy A., Chantalat S., Sudharshan R., Papili Gao N., Stockholm D., Corre G., Fourel G., Deleuze J.F., Gunawan R, & Paldi A. (2022). Global genome decompaction leads to stochastic activation of gene expression as a first step toward fate commitment in human hematopoietic cells. PLOS Biology, 20(10), e3001849.

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2

AML Cell Lines and Cord Blood CD34+ Cells

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The AML cell lines U937, HL-60, and Molm-14 were cultured in RPMI media (Life Technologies) with 10% FBS in a Thermo Scientific cell culture incubator at 5% CO₂. For Molm-14 xenografts, Molm-14 cells were cultured at 1% or 21% O₂ in equal numbers before engraftment. Cell lines were obtained as previously reported15 (link); Molm-14 were authenticated by PCR for characteristic FLT3-ITD prior to engraftment. CD34+ cells were purified from cord blood using MACS cell separation (Miltenyi Biotec) and cultured in serum-free expansion media (StemCell Technologies) supplemented with 100 U/ml penicillin/streptomycin, 40 ng/ml Flt-3, 25 ng/ml SCF, and 50 ng/ml TPO (Miltenyi Biotec). Primary murine stromal cells were BM stromal cells were obtained by culturing whole BM cells isolated from the experimental animals in IMDM (Life Technologies) with 20% Vesicle-free FBS15 (link) for 4 days, then washing 2x with versene.

Hornick N.I., Huan J., Doron B., Goloviznina N.A., Lapidus J., Chang B.H, & Kurre P. (2015). Serum Exosome MicroRNA as a Minimally-Invasive Early Biomarker of AML. Scientific Reports, 5, 11295.

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3

Myelination of Rat and Mouse DRGs

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DRGs were excised from E14 Sprague-Dawley (SD) rat or E12 mouse embryos. The entire ganglia were plated on 24 well plates coated with poly-L lysine (PLL) (Sigma Aldrich) and cultrex mouse laminin I (R&D systems), and cultured with MACS ^R Neuro Medium (Miltenyi Biotec) supplemented with 10% fetal bovine serum (FBS) (Sigma Aldrich), 1:200 Glutamax (Thermo Fischer Scientific), 50 U/ml Penicillin/Streptomycin (Thermo Fischer Scientific) and 100ng/ml 2.5S nerve growth factor (NGF) (COSMO BIO). Sixteen hours later, the medium was changed to MACS ^R Neuro Medium supplemented with 1% MACS^R NeuroBrew^R-B21 (Miltenyi Biotec), 1:200 Glutamax, 50 U/ml Penicillin/Streptomycin and 100 ng/ml 2.5S NGF. At day 5, the medium was replaced DMEM supplemented with 10% FBS, 1:200 Glutamax, 50 U/ml Penicillin/Streptomycin and 100 ng/ml 2.5S NGF, and thereafter half of the medium was changed every other day. On day14, 50 μg/ml of L-ascorbic acid (Sigma Aldrich) was added to the medium to initiate myelination. For EM analysis, DRGs were cultured on Cell Disk LF (Sumitomo Bakelite Co.) coated PLL and cultrex mouse laminin I.

Numata-Uematasu Y., Wakatsuki S., Kobayashi-Ujiie Y., Sakai K., Ichinohe N, & Araki T. (2023). In vitro myelination using explant culture of dorsal root ganglia: An efficient tool for analyzing peripheral nerve differentiation and disease modeling. PLOS ONE, 18(5), e0285897.

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4

CD8+ T Cell CAR Transduction

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FACS-purified CD8⁺ T cell subsets were stimulated with a MACS-GMP T Cell TransAct Cocktail (Miltenyi Biotec). Stimulated cells were transduced the following day with a bidirectional lentiviral vector encoding a CD19-specific CAR with a CD28 costimulus in sense and the LNGFR marker gene in antisense and then cultured for 13 d in TexMACS Medium (Miltenyi Biotec) supplemented with 3% FBS, 1% penicillin/streptomycin, IL-7 (25 U/ml, Miltenyi Biotec), and IL-15 (50 U/ml, Miltenyi Biotec). Magnetically purified CD3⁺ cells were processed similarly for control purposes.

Galletti G., De Simone G., Mazza E.M., Puccio S., Mezzanotte C., Bi T.M., Davydov A.N., Metsger M., Scamardella E., Alvisi G., De Paoli F., Zanon V., Scarpa A., Camisa B., Colombo F.S., Anselmo A., Peano C., Polletti S., Mavilio D., Gattinoni L., Boi S.K., Youngblood B.A., Jones R.E., Baird D.M., Gostick E., Llewellyn-Lacey S., Ladell K., Price D.A., Chudakov D.M., Newell E.W., Casucci M, & Lugli E. (2020). Two subsets of stem-like CD8+ memory T cell progenitors with distinct fate commitments in humans. Nature immunology, 21(12), 1552-1562.

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CD8+ T Cell CAR Transduction

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FACS-purified CD8⁺ T cell subsets were stimulated with a MACS-GMP T Cell TransAct Cocktail (Miltenyi Biotec). Stimulated cells were transduced the following day with a bidirectional lentiviral vector encoding a CD19-specific CAR with a CD28 costimulus in sense and the LNGFR marker gene in antisense and then cultured for 13 d in TexMACS Medium (Miltenyi Biotec) supplemented with 3% FBS, 1% penicillin/streptomycin, IL-7 (25 U/ml, Miltenyi Biotec), and IL-15 (50 U/ml, Miltenyi Biotec). Magnetically purified CD3⁺ cells were processed similarly for control purposes.

Galletti G., De Simone G., Mazza E.M., Puccio S., Mezzanotte C., Bi T.M., Davydov A.N., Metsger M., Scamardella E., Alvisi G., De Paoli F., Zanon V., Scarpa A., Camisa B., Colombo F.S., Anselmo A., Peano C., Polletti S., Mavilio D., Gattinoni L., Boi S.K., Youngblood B.A., Jones R.E., Baird D.M., Gostick E., Llewellyn-Lacey S., Ladell K., Price D.A., Chudakov D.M., Newell E.W., Casucci M, & Lugli E. (2020). Two subsets of stem-like CD8+ memory T cell progenitors with distinct fate commitments in humans. Nature immunology, 21(12), 1552-1562.

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6

Isolation and Culture of NK Cells

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PBMCs were supplied commercially (AllCells) and magnetically separated with NK Cell Isolation Kit (Miltenyi Biotec). Upon isolation, NK cells were cultured in complete NK medium, prepared with NK MACS media (Miltenyi Biotec) supplemented with human serum (1:20), penicillin streptomycin (1:100), NK supplement (1:100, Miltenyi Biotec), and IL-2 (500 IU/ml). Cells were transferred into a 24-well G-REX (Wilson Wolf) five days after isolation, and fresh NK media was then added every 2–3 days to ensure cell health. Purity was measured by flow cytometry using Miltenyi Biotec’s CD56 REAfinity (PE-Vio770; 1:50) at day nine of the NK cell culture. Cells were used for transfection 15–18 days after isolation.

Loo J., Sicher I., Goff A., Kim O., Clary N., Alexeev A., Sulchek T., Zamarayeva A., Han S, & Calero-Garcia M. (2021). Microfluidic transfection of mRNA into human primary lymphocytes and hematopoietic stem and progenitor cells using ultra-fast physical deformations. Scientific Reports, 11, 21407.

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7

Generation and Characterization of M2 Macrophages

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The human U87 cells line and human umbilical vein endothelial cells line (HUVECs) were obtained from American Type Culture Collection (ATCC). U87 cells were grown in high glucose Dulbecco's Modified Eagle Medium (DMEM) (Gibco, Invitrogen) supplemented with 10% FBS (Gibco, Invitrogen) and 1% penicillin/streptomycin (Gibco, Invitrogen). HUVECs were maintained in RPMI 1640 (Gibco, Invitrogen) supplemented with 10% FBS and 1% penicillin/streptomycin. All the cells were grew in 5% CO₂ at 37°C.
Fresh human peripheral blood was obtained from Sun Yat-sen Memorial Hospital, according to the guidelines approved by the ethics committee at Sun Yat-sen Memorial Hospital. Monocyte-derived M₂ macrophages were generated from human PBMC as described previously[12 (link)]. Briefly, PBMC were isolated from peripheral blood by density gradient centrifugation. After being purified by using anti-CD14 microbeads (Miltenyi Biotec), monocytes were incubated in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin for 3 days. M₂ macrophage polarization were obtained by removing the median and culturing cells for another 3 days in DMEM supplemented with 40 ng/ml Recombinant Human IL-4 (PeproTech).

Rong X., Huang B., Qiu S., Li X., He L, & Peng Y. (2016). Tumor-associated macrophages induce vasculogenic mimicry of glioblastoma multiforme through cyclooxygenase-2 activation. Oncotarget, 7(51), 83976-83986.

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8

Generating Mixed Glial Cells from Mouse Brains

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Mixed glial cells were generated from male and female P2-7 C57BL/6 pups according to the protocol of the Neural Tissue Dissociation Kit (P) (Miltenyi Biotec). In short, brains were dissected, cerebellum and meninges were removed and tissue was dissociated via mechanical and papain enzyme digestion prior to filtration through a 40 μm strainer. Cells were plated in poly-L-lysine-coated (10 μg/ml, Sigma) 96 well flat, glass-bottomed plates (BD Falcon) at a density of 10⁵ cells per well and cultures were maintained at 37°C, 5% CO₂. Cells were cultured for 5 days in DMEM (Life Technologies) supplemented with PDGFαα (10 ng/ml; PeproTech), 10% endotoxin-free FCS, 1% penicillin/streptomycin and 1% L-glutamine (Life Technologies). Cells were cultured for a further 2 days in neural medium (Miltenyi Biotec) supplemented with 1% penicillin/streptomycin, 1% L-glutamine, 2% B27/MACS Neuro Brew21 (Miltenyi Biotec) and PDGFαα (10 ng/ml; PeproTech). At day 7 in culture, PDGFαα was withdrawn to allow oligodendrocyte differentiation and cells were stimulated with 5% T_reg-conditioned media, rCCN3, anti-CCN3 (clone 231216) or isotype control (clone 54447) (all R&D Systems) or controls for up to 5 days.

Dombrowski Y., O’Hagan T., Dittmer M., Penalva R., Mayoral S.R., Bankhead P., Fleville S., Eleftheriadis G., Zhao C., Naughton M., Hassan R., Moffat J., Falconer J., Boyd A., Hamilton P., Allen I.V., Kissenpfennig A., Moynagh P.N., Evergren E., Perbal B., Williams A.C., Ingram R.J., Chan J.R., Franklin R.J, & Fitzgerald D.C. (2017). Regulatory T cells promote myelin regeneration in the Central Nervous System. Nature neuroscience, 20(5), 674-680.

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Generating Mixed Glial Cells from Mouse Brains

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Mixed glial cells were generated from male and female P2-7 C57BL/6 pups according to the protocol of the Neural Tissue Dissociation Kit (P) (Miltenyi Biotec). In short, brains were dissected, cerebellum and meninges were removed and tissue was dissociated via mechanical and papain enzyme digestion prior to filtration through a 40 μm strainer. Cells were plated in poly-L-lysine-coated (10 μg/ml, Sigma) 96 well flat, glass-bottomed plates (BD Falcon) at a density of 10⁵ cells per well and cultures were maintained at 37°C, 5% CO₂. Cells were cultured for 5 days in DMEM (Life Technologies) supplemented with PDGFαα (10 ng/ml; PeproTech), 10% endotoxin-free FCS, 1% penicillin/streptomycin and 1% L-glutamine (Life Technologies). Cells were cultured for a further 2 days in neural medium (Miltenyi Biotec) supplemented with 1% penicillin/streptomycin, 1% L-glutamine, 2% B27/MACS Neuro Brew21 (Miltenyi Biotec) and PDGFαα (10 ng/ml; PeproTech). At day 7 in culture, PDGFαα was withdrawn to allow oligodendrocyte differentiation and cells were stimulated with 5% T_reg-conditioned media, rCCN3, anti-CCN3 (clone 231216) or isotype control (clone 54447) (all R&D Systems) or controls for up to 5 days.

Dombrowski Y., O’Hagan T., Dittmer M., Penalva R., Mayoral S.R., Bankhead P., Fleville S., Eleftheriadis G., Zhao C., Naughton M., Hassan R., Moffat J., Falconer J., Boyd A., Hamilton P., Allen I.V., Kissenpfennig A., Moynagh P.N., Evergren E., Perbal B., Williams A.C., Ingram R.J., Chan J.R., Franklin R.J, & Fitzgerald D.C. (2017). Regulatory T cells promote myelin regeneration in the Central Nervous System. Nature neuroscience, 20(5), 674-680.

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Macrophage Differentiation and Treatment

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Raw 264.7 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; GIBCO BRL, Life Technologies, Inc., Rockville, MD, USA) with 10% fetal bovine serum (FBS; GIBCO BRL, Life Technologies, Inc., Rockville, MD, USA) and penicillin/streptomycin (100 U/ml and 100 U/ml, respectively; Sigma-Aldrich) at 37 °C in a 5% CO2 incubator. Cells were treated with LPS (1 µg/ml), IFN-γ (50 ng/ml), H₂O₂ (10 µmol/L), Oligo (10 µg/mL), NAC (1 mmol/L), visomitin (20 nmol/L), 3PO (10 µmol/L) and mitoquinone (50 nm). Bone marrow-derived cell from C57BL/6 mice were cultured in DMEM media supplemented with 10% FBS and 1% penicillin/streptomycin and differentiated to bone marrow-derived macrophages (BMDMs) by recombinant murine granulocyte- macrophage colony stimulating factor (GM-CSF) (25 ng/mL; Miltenyi Biotech) for 7 days.

Huang W., Wang L., Huang Z., Sun Z, & Zheng B. (2024). Peroxiredoxin 3 has a crucial role in the macrophage polarization by regulating mitochondrial homeostasis. Respiratory Research, 25, 110.

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Penicillin streptomycin

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27 protocols using penicillin streptomycin

Isolation and Expansion of Human CD34+ Cells

AML Cell Lines and Cord Blood CD34+ Cells

Myelination of Rat and Mouse DRGs

CD8+ T Cell CAR Transduction

CD8+ T Cell CAR Transduction

Isolation and Culture of NK Cells

Generation and Characterization of M2 Macrophages

Generating Mixed Glial Cells from Mouse Brains

Generating Mixed Glial Cells from Mouse Brains

Macrophage Differentiation and Treatment

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