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Pen strep

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Pen/Strep is a sterile solution containing the antibiotics penicillin and streptomycin. It is commonly used in cell culture applications to prevent bacterial contamination.

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Lab products found in correlation

Gm csf, by R&D Systems (2 mentions) Matrigel, by BD (2 mentions) Glutamax, by Thermo Fisher Scientific (2 mentions) Epidermal growth factor (egf), by R&D Systems (2 mentions) Insulin, by Merck Group (2 mentions) Lymphoprep, by STEMCELL (2 mentions) Glutamine, by R&D Systems (2 mentions) Ab1927, by Abcam (2 mentions) Atplite 1step assay, by PerkinElmer (2 mentions) Fbs6450, by STEMCELL (2 mentions) Hflt3l, by STEMCELL (2 mentions) Rpmi 1640, by Thermo Fisher Scientific (2 mentions) Recombinant human gm csf, by R&D Systems (1 mentions) Recombinant human il 4, by R&D Systems (1 mentions) T cell isolation kit 2, by Miltenyi Biotec (1 mentions) Ficoll paque, by GE Healthcare (1 mentions) Sodium pyruvate, by R&D Systems (1 mentions) Anti cd14 microbeads, by Miltenyi Biotec (1 mentions) Holotransferrin, by R&D Systems (1 mentions) Rpmi 1640, by Sartorius (1 mentions)

17 protocols using pen strep

1

Monocyte-Derived Dendritic Cell Generation

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Fifty to 70 ml of blood was taken by venous puncture. Peripheral blood mononuclear cells were isolated from heparinized blood by Ficoll-Paque (GE Healthcare) purification. Briefly, blood was diluted 1:1 with sodium chloride and tubes were centrifuged at 400g for 30 minutes with the brake off. Cells were harvested from the interface of the Ficoll layer, and were washed and enumerated. Monocytes were isolated using anti-CD14 microbeads, according to the manufacturer’s instruction (Miltenyi Biotec, San Diego, CA, USA). Cells were cultured in RPMI 1640 with 10% human serum and L-glutamine, pen/strep, non-essential amino acids, sodium pyruvate, 2-ME, 40 ng/ml of recombinant human IL-4 and 40 ng/ml of recombinant human GM-CSF (both from R&D Systems). Cytokines were replenished on days 3 and 6, and cells were used on day 7. For some experiments, CD4+ T cells were isolated from the CD14- fraction using the T cell isolation II kit (Miltenyi Biotec). Cells were then cultured with MoDCs in the presence or absence or RSV. At 48 hours, RNA was extracted and message levels of IFN-γ, IL-5 and IL-13 were determined by qPCR.
Ptaschinski C., Mukherjee S., Moore M.L., Albert M., Helin K., Kunkel S.L, & Lukacs N.W. (2015). RSV-Induced H3K4 Demethylase KDM5B Leads to Regulation of Dendritic Cell-Derived Innate Cytokines and Exacerbates Pathogenesis In Vivo. PLoS Pathogens, 11(6), e1004978.
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2

Erythropoietin-Stimulated Murine Bone Marrow Cells

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CD71+ murine bone marrow cells were erythropoietin stimulated for two days in DME with 10% fetal calf serum, 1% pen-strep, SCF, holotransferrin (R&D Systems, Minneapolis, MN). Lysates (30 μg) of CD71+ cells were separated by 10% SDS-PAGE, transferred to nitrocellulose, and serially probed with Fanconi C and GAPDH antibodies (31 (link)). Three independent lysates were analyzed, and representative blots shown.
Stat3 and pY705-Stat3 were quantified by ELISA (Cell Signaling Technology, Danvers, MA). Three independent lysates were analyzed in duplicate.
Hasan S., Hu L., Williams O, & Eklund E.A. (2022). Ruxolitinib ameliorates progressive anemia and improves survival during episodes of emergency granulopoiesis in Fanconi C−/− mice. Experimental hematology, 109, 55-67.e2.
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3

Differentiation of primary human macrophages

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Primary human macrophages were differentiated from peripheral blood monocytes cells (PBMC). After isolation of PBMC’s in a density gradient medium, cells were split into 12-well culture plates (Greiner CELLSTAR multiwell culture plates, 0.4 × 106 cells/well) and incubated at 37 °C, 5% v/v CO2 and 95% v/v O2 for 1 h in a free serum medium (RPMI 1640, 2 mM l-glutamine, 1% Pen/Strep, Biological Industries). Non-adherent contaminated cells were removed and remaining monocytes differentiated to human microglia like cells for 10 days with recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF, 10 ng/ml; R&D systems) in a full RPMI medium (RPMI 1640, 10% FBS, 2 mM l-glutamine, 1% Pen/Strep) and incubated at 37 °C, 5% v/v CO2 and 95% v/v O2.
Fassler M., Rappaport M.S., Cuño C.B, & George J. (2021). Engagement of TREM2 by a novel monoclonal antibody induces activation of microglia and improves cognitive function in Alzheimer’s disease models. Journal of Neuroinflammation, 18, 19.
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4

Isolation and Transduction of Murine CML-LSC

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Animal studies were performed according to a protocol approved by the Animal Care and Use Committees of Northwestern University and Jesse Brown VA Medical Center. Bone marrow mononuclear cells were obtained from the femurs of Wt or Icsbp−/− C57/BL6 mice. Granulocyte/monocyte progenitor cells were cultured (2 × 105 cells per ml) in DME media supplemented with 10% fetal calf serum, 1% pen-strep, 10 ng/ml murine GM-CSF (R & D Systems Inc., Minneapolis, MN), 10 ng/ml murine recombinant IL-3 (R & D Systems Inc.) and 100 ng/ml of murine recombinant stem cell factor (Scf: R & D Systems Inc.) (referred to as myeloid progenitor cells in these studies). CD34+ cells were separated from the cultures using the Miltenyi magnetic bead system for extraction of RNA or proteins. These cells represent the CML-LSC in murine models during CP [43 (link)]. For some experiments, cells were transduced with retroviral vectors to express p210 Bcr-abl (in MiGR1 vector), DN-Gas2 (in MSCVpuro vector), or DN-Shp2 (in MSCVneo vector). Replication incompetent retroviral vectors were generated using the Ecopack packaging plasmid, as described [23 (link)].
Hjort E.E., Huang W., Hu L, & Eklund E.A. (2016). Bcr-abl regulates Stat5 through Shp2, the interferon consensus sequence binding protein (Icsbp/Irf8), growth arrest specific 2 (Gas2) and calpain. Oncotarget, 7(47), 77635-77650.
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5

Isolation of Monocyte Precursors from Bone Marrow

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The monocyte precursors were isolated from mice or rat bone marrow of the hind limbs by centrifugation of the bone marrow cell mix through a percoll (GE Healthcare, 17-0891-02) density gradient (3 ml 45%, 5 ml 62% and 3 ml 81% (v/v) percoll in HBSS). The cell fraction between 45 and 62% layers containing monocyte precursors was cultured in IMDM media (21056-023, Gibco by life technologies) containing 20% (v/v) FCS, 1% (v/v) Pen/Strep and 100 ng/ml M-CSF (R&D Systems) at 37 °C, 5% CO2.
Funk N., Munz M., Ott T., Brockmann K., Wenninger-Weinzierl A., Kühn R., Vogt-Weisenhorn D., Giesert F., Wurst W., Gasser T, & Biskup S. (2019). The Parkinson’s disease-linked Leucine-rich repeat kinase 2 (LRRK2) is required for insulin-stimulated translocation of GLUT4. Scientific Reports, 9, 4515.
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6

Derivation and Maintenance of Intestinal Organoids

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Derivation and maintenance of HIOs followed published protocols1 (link), 25 (link). Briefly, HIOs were embedded in Matrigel (BD Biosciences) and overlaid with Advanced DMEM-F12 medium (Invitrogen, Carlsbad, CA) containing 1X B27 supplement (Invitrogen), 1X GlutaMAX (Life Technologies, Carlsbad, CA), 10 µM Hepes, 10% pen/strep, 100 ng/mL rhNoggin (R&D Systems), 100 ng/mL epidermal growth factor (R&D Systems), and approximately 500 ng/mL R-Spondin1 (RSPO1). RSPO1 was obtained from conditioned media collected from a HEK293 cell line that was stably transfected and zeocin-selected for the RSPO1 expression vector. Media was changed every two to four days, and HIOs were transferred to fresh Matrigel once a week until they reached approximately 2 to 3 mm in diameter for experiments. This size was reached on average 48 days after initial spheroid formation.
Sidar B., Jenkins B.R., Huang S., Spence J.R., Walk S.T, & Wilking J.N. (2019). Long-Term Flow through Human Intestinal Organoids with the Gut Organoid Flow Chip (GOFlowChip). Lab on a chip, 19(20), 3552-3562.
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7

MLL-FP Transduced Leukemia Model

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Kit+ bone marrow cells from 5-FU primed mice were enriched by Hematopoietic Stem and Progenitor Cell Enrichment Kit (Stem Cell Technologies) and transduced with freshly packaged retroviruses expressing MLL-FPs. Cells were selected with 1mg/ml neomycin for 1 week with refreshed antibiotics every 3 days. Established mouse leukemia cell lines were maintained in IMDM+15%FBS (for mouse myeloid long-term cultures, Stem Cell Technologies)+1%Pen/Strep+10ng/ml recombinant murine IL3 (R&D Systems).
Chen L., Sun Y., Wang J., Jiang H, & Muntean A.G. (2016). Differential regulation of the c-Myc/Lin28 axis discriminates subclasses of rearranged MLL leukemia. Oncotarget, 7(18), 25208-25223.
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8

Evaluating Compound Effects on T-ALL Proliferation

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Ba/F3 cells were seeded in triplicate into 96-well plates (1x104cells/well) and treated with indicated compound concentrations or vehicle (DMSO). Proliferation was assessed using the ATPlite 1step assay (PerkinElmer). To calculate relative proliferation, luminescence values at the start of the drug incubation were subtracted from the values after 24 hours of drug incubation and normalized to the DMSO control.
For T-ALL xenograft experiments, viably frozen xenograft cells were thawed and incubated overnight in xenograft MEMα medium containing 10% FBS6450 (Stemcell Technologies), 10% human AB+ serum (Institut de Biotechnologies Jacques BOY), Pen/Strep, Glutamine, 10ng/mL hIL7 (R&D systems), 50ng/ml hSCF and 20ng/ml hFLT3L (Stemcell Technologies) and 20nM insulin (Sigma). The next day, cells were passed through Lymphoprep (Stemcell Technologies) and plated in triplicate in 96-well plates previously coated with 10µg/ml anti-Fc (ab1927, Abcam) followed by overnight coating with 2µg/ml mDL4-Fc peptide (VIB PSF). Plating was done in drug containing xenograft medium at 1x105cells/well. After 72 hrs, relative cell numbers were measured using the ATPlite 1step assay (PerkinElmer) and apoptosis was determined by flow cytometry analysis of Annexin V expression.
Girardi T., Vereecke S., Sulima S.O., Khan Y., Fancello L., Briggs J.W., Schwab C., de Beeck J.O., Verbeeck J., Royaert J., Geerdens E., Vicente C., Bornschein S., Harrison C.J., Meijerink J.P., Cools J., Dinman J.D., Kampen K.R, & De Keersmaecker K. (2017). The T-cell leukemia associated ribosomal RPL10 R98S mutation enhances JAK-STAT signaling. Leukemia, 32(3), 809-819.
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9

Evaluating Compound Effects on T-ALL Proliferation

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Ba/F3 cells were seeded in triplicate into 96-well plates (1x104cells/well) and treated with indicated compound concentrations or vehicle (DMSO). Proliferation was assessed using the ATPlite 1step assay (PerkinElmer). To calculate relative proliferation, luminescence values at the start of the drug incubation were subtracted from the values after 24 hours of drug incubation and normalized to the DMSO control.
For T-ALL xenograft experiments, viably frozen xenograft cells were thawed and incubated overnight in xenograft MEMα medium containing 10% FBS6450 (Stemcell Technologies), 10% human AB+ serum (Institut de Biotechnologies Jacques BOY), Pen/Strep, Glutamine, 10ng/mL hIL7 (R&D systems), 50ng/ml hSCF and 20ng/ml hFLT3L (Stemcell Technologies) and 20nM insulin (Sigma). The next day, cells were passed through Lymphoprep (Stemcell Technologies) and plated in triplicate in 96-well plates previously coated with 10µg/ml anti-Fc (ab1927, Abcam) followed by overnight coating with 2µg/ml mDL4-Fc peptide (VIB PSF). Plating was done in drug containing xenograft medium at 1x105cells/well. After 72 hrs, relative cell numbers were measured using the ATPlite 1step assay (PerkinElmer) and apoptosis was determined by flow cytometry analysis of Annexin V expression.
Girardi T., Vereecke S., Sulima S.O., Khan Y., Fancello L., Briggs J.W., Schwab C., de Beeck J.O., Verbeeck J., Royaert J., Geerdens E., Vicente C., Bornschein S., Harrison C.J., Meijerink J.P., Cools J., Dinman J.D., Kampen K.R, & De Keersmaecker K. (2017). The T-cell leukemia associated ribosomal RPL10 R98S mutation enhances JAK-STAT signaling. Leukemia, 32(3), 809-819.
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10

Isolation and Differentiation of Myeloid Progenitor Cells

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Mononuclear cells were obtained from the femurs of Wt C57/BL6 mice. Lin−Sca1+ cells were separated using the Miltenyi magnetic bead system (according to manufacturer’s instructions; Miltenyi Biotechnology, Auburn, CA). Cells were cultured for 48 hrs in DME media supplemented with 10% fetal calf serum, 1% pen-strep, and murine recombinant GM-CSF (20 ng/ml), Scf (100 ng/ml) and IL-3 (10 ng/ml) (R & D Systems Inc., Minneapolis, MN) (2 × 105 cells per ml). Some CD34+ cells were separated for analysis (using the Miltenyi magnetic bead system); referred to as myeloid progenitor cells in this study. Other cells were differentiated after 24 hrs with G-CSF or IL1β, as described (9 (link),30 (link)).
Wang H., Bei L., Shah C.A., Hu L, & Eklund E.A. (2015). HoxA10 Terminates Emergency Granulopoiesis by Increasing Expression of Triad. Journal of immunology (Baltimore, Md. : 1950), 194(11), 5375-5387.
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