Pen strep

Manufactured by BioLegend

Pen-Strep is a sterile solution containing penicillin and streptomycin, two commonly used antibiotics. It is often used in cell culture applications to prevent bacterial contamination.

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Lab products found in correlation

Facsymphony a5, by BD (2 mentions) Stem cell factor (scf), by BioLegend (2 mentions) Polybrene, by Merck Group (1 mentions) Mouse il 6, by BioLegend (1 mentions) X vivo 10, by Lonza (1 mentions) Mouse il 3, by BioLegend (1 mentions) Annexin a5 apoptosis detection kit, by BioLegend (1 mentions) Hct116, by Thermo Fisher Scientific (1 mentions) Ebm 2 basal medium, by Lonza (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions) Rpmi 1640, by American Type Culture Collection (1 mentions) Penicillin streptomycin, by Bio-Techne (1 mentions) Antibiotic antimycotic, by Thermo Fisher Scientific (1 mentions) Huvecs, by Lonza (1 mentions) Hct116, by Lonza (1 mentions) Trypsin edta, by Bio-Techne (1 mentions) Ls separation column, by Miltenyi Biotec (1 mentions) Cd14 microbead, by Miltenyi Biotec (1 mentions) Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (1 mentions)

8 protocols using pen strep

Generation of Antigen-Specific T Cell Retrogenic Mice

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SP33rg mice were generated as described previously (Leonard et al., 2017 (link); McDonald et al., 2015 (link)). In brief, the SP33 TCRα was cloned into a retroviral construct modified from Turner and colleagues (McDonald et al., 2015 (link); Turner et al., 2010 (link)). Plat-E cells, also previously described (Morita et al., 2000 (link)), were used to generate retrovirus. TCRα^−/− CD4-Cre⁺ TCRβtg⁺ mice on a B6 background were injected with 5-fluorouracil (APP Pharmaceuticals) 3 d before bone marrow harvest. Bone marrow cells were cultured for 2 d in X-VIVO 10 (Lonza) containing 15% FCS, 1% pen/strep, 100 ng/ml mouse stem cell factor, 10 ng/ml mouse IL-3, and 20 ng/ml mouse IL-6 (BioLegend). Cells were infected with retrovirus by spinfection in the presence of 6 µg/ml polybrene (EMD Millipore) and cultured for an additional 24 h. All spinfected cells were then mixed with 5 × 10⁶ freshly harvested bone marrow ‘‘filler’’ cells from Rag1^−/− mice and injected into lethally irradiated (900 rads) CD45^.1/.1B6.SJL recipient mice to generate SP33rg mice. SP33rg cells were isolated from retrogenic mice 6–8 wk after bone marrow reconstitution. CD4⁺ T cells were FACS–purified from SP33rg mice following CD4 MACS (Miltenyi Biotec) enrichment and staining with the following antibodies: anti-CD8b (Ly-3), anti-CD45.1 (A20), anti-CD45.2 (104), and anti-Thy1.1 (OX-7).

Klawon D.E., Gilmore D.C., Leonard J.D., Miller C.H., Chao J.L., Walker M.T., Duncombe R.K., Tung K.S., Adams E.J, & Savage P.A. (2021). Altered selection on a single self-ligand promotes susceptibility to organ-specific T cell infiltration. The Journal of Experimental Medicine, 218(6), e20200701.

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Apoptosis Assay of LT-HSCs

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Staining for apoptosis was performed using Annexin V and 7-AAD. As defined by SLAM markers, 1,000 LT-HSCs were sorted directly into 96 well plates containing SFEMII with Pen-Strep and SCF (100 ng/ml), TPO (50 ng/ul), with or without IGF1 (100 ng/ml) (BioLegend, Peprotech and StemCell Technologies) for 18h and 40h at 37°C and 5% CO₂. Cells were stained with Annexin V and 7-AAD (Annexin A5 Apoptosis Detection Kit, Biolegend). Cells were collected and analyzed by flow cytometry on a FACSymphony A5 (BD).

Young K., Eudy E., Bell R., Loberg M.A., Stearns T., Sharma D., Velten L., Haas S., Filippi M.D, & Trowbridge J.J. (2021). Decline in IGF1 in the Bone Marrow Microenvironment Initiates Hematopoietic Stem Cell Aging. Cell stem cell, 28(8), 1473-1482.e7.

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Mitochondrial Function in LT-HSCs

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Staining for mitochondrial function was performed as previously described(Hinge et al., 2020 (link)). As defined by SLAM markers, 1,000 LT-HSCs were sorted directly into 96 well plates containing SFEMII with Pen-Strep and SCF (100 ng/ml), TPO (50 ng/ul), with and without IGF1 (100 ng/ml), (BioLegend, Peprotech and StemCell Technologies) for 18h at 37°C and 5% CO₂. LT-HSCs were washed and incubated at 37°C and 5% CO₂ for 30min with TMRE (0.1μM to measure mitochondrial membrane potential, Sigma). Cells were collected and analyzed by flow cytometry on a FACSymphony A5 (BD) within 60min of mitochondrial staining.

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Quantifying Mitochondrial Dynamics in LT-HSCs

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As defined by SLAM markers, 100 LT-HSCs from young and middle-aged mito-Dendra2 mice were sorted directly into 96-well plates containing SFEMII with Pen-Strep and SCF (100 ng/ml), TPO (50 ng/ul), with or without IGF1 (100 ng/ml) (BioLegend, Peprotech and StemCell Technologies) for 18h at 37°C and 5% CO₂. Cells were imaged live at high resolution Nyquist limit setting (0.1um XY pixel size) at 60X magnification using 1.2 AU pinhole to give an optical section thickness of 0.39 um. Z-stacks were acquired with a step size of 0.1 um. Typically, 100–120 Z sections were taken. Deconvolution analysis of the images was done using NIS elements. Scale bars in images represent 5μm.

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Culturing Human Cell Lines for Research

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Human umbilical
vein endothelial primary cells (HUVECs) and human colorectal carcinoma
cell lines (HCT116) were purchased from Lonza and the American Type
Culture Collection (ATCC), respectively. HUVECs were cultured in EBM-2
Basal Medium (Lonza), supplied with Microvascular Endothelial Cell
Growth Medium SingleQuots supplements (EGM-2 MV, Lonza) and 1% Antibiotic-Antimycotic
(Gibco). HUVECs from passages 3–8 were used in the experiments.
The HCT116 were cultured in Dulbecco’s modified eagle medium
(DMEM, Gibco), supplied with 10% fetal bovine serum (FBS, Gibco) and
1% penicillin-streptomycin (Bio-Techne Corporation). The T cells were
obtained from the Human Immunology Core of Penn Medicine and maintained
in RPMI-1640 (ATCC), supplied with 10% FBS, 1% Pen-Strep, and 10 ng/mL
Recombinant Human IL-2 (Biolegend). All of the cells were maintained
regularly with medium change each other day. For HUVECs and HCT116,
0.05% Trypsin-EDTA (Bio-Techne Corp.) was employed for the passaging.

Zhao Y., Wu Y., Islam K., Paul R., Zhou Y., Qin X., Li Q, & Liu Y. (2024). Microphysiologically Engineered Vessel-Tumor Model to Investigate Vascular Transport Dynamics of Immune Cells. ACS Applied Materials & Interfaces, 16(18), 22839-22849.

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Isolation and Differentiation of Human Macrophages

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Human peripheral blood mononuclear cells (PBMCs) were obtained from leukopaks. Briefly, whole blood from leukopaks was mixed with 1× PBS, and Ficoll-Hypaque solution (Cytiva) was carefully laid underneath of the blood and PBS mixture. Cells were centrifuged at 400g for 30 min with no break. After the centrifugation, cells in the buffy coat were collected and lysed with ACK lysis buffer (Gibco) on ice for 5 min and washed with excess amounts of RPMI (Gibco) to obtain PBMCs. CD14⁺ monocytes were isolated (>95% purity; Extended Data Fig. 4g) from the PBMCs by using CD14 MicroBeads (Miltenyi Biotec) following the manufacturer’s instructions. One hundred million cells were incubated with 200 μl of CD14 MicroBeads for 15 min at 4 °C, washed with MACS buffer and loaded onto an LS separation column (Miltenyi Biotec). CD14⁺ cells were eluted from the column after three washes with MACS buffer. The purity of the monocytes was evaluated via flow cytometry.
Human macrophages were differentiated from the monocytes. Briefly, CD14⁺ monocytes were cultured in RPMI-1640 supplemented with 10% FBS, 100 U ml⁻¹ pen–strep and 50 ng ml⁻¹ human macrophage colony-stimulating factor (M-CSF; BioLegend) at 37 °C with 5% CO₂ for 7 d. Medium was refreshed on day 4, and before signaling assays, human macrophages were starved in growth medium overnight with 2% FBS.

Krüger S., Brandt E., Klinger M., Krüger S, & Kreft B. (2000). Interleukin-8 Secretion of Cortical Tubular Epithelial Cells Is Directed to the Basolateral Environment and Is Not Enhanced by Apical Exposure to Escherichia coli. Infection and Immunity, 68(1), 328-334.

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Generation of Mouse Bone Marrow-Derived Macrophages

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Mouse BMDMs were differentiated from monocytes in the bone marrow. Briefly, femurs and tibias were dissected from both legs of a C57BL/6 mouse, and one end of each bone was cut open with a pair of scissors. Bone marrow was obtained by centrifuging bones with open ends at 2,000g for 30 s. The collected bone marrow was resuspended and cultured in DMEM supplemented with 10% FBS, 100 U ml⁻¹ pen–strep, 2 mM l-glutamine and 100 ng ml⁻¹ mouse M-CSF (BioLegend) for 7 d at 37 °C with 5% CO₂ to obtain mouse macrophages. Medium was refreshed on day 4, and before signaling assays, mouse macrophages were starved in growth medium overnight with 2% FBS.

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Evaluating NK Cell Degranulation

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Commercially available buffy coats were acquired from Versiti Indiana Blood Center. Human peripheral blood mononuclear cells (PBMC) were isolated using Ficoll-Paque Plus (GE-Healthcare, Pittsburgh, PA) gradient centrifugation and cultured in RPMI1640 with 10% FBS, 1% pen/strep, and 20 ng/mL rhIL-2 (~ 41.2 U/mL, BioLegend) at 37 °C in a CO₂ incubator for 5 days. 5 × 10⁴ WT, TKO, and 5GKO ipLDECs were plated in 48-well plates in duplicate or triplicate one day ahead of co-culture. 5 × 10⁵ PBMC were added to porcine cells at an E:T ratio of 10:1, and co-cultured for 2 h at 37 °C in a CO₂ incubator. Then cells were collected and stained with fluorochrome-conjugated antibodies against human CD45, CD3, CD56, CD107a (BioLegend), and Invitrogen eBioscience Fixable Viability Dye eFluor 780 (Thermo Fisher Scientific). Stained cells were fixed with 2% PFA and subsequently analyzed using a LSR4 flow cytometer (BD Biosciences). After pre-gating on CD45⁺ live singlets, NK cell degranulation activity was assessed based on percentage and mean fluorescence intensity (MFI) of CD107a in a CD3^-CD56⁺ cell population. Flow data were analyzed using FlowJo v10 software (BD Biosciences).

Li P., Walsh J.R., Lopez K., Isidan A., Zhang W., Chen A.M., Goggins W.C., Higgins N.G., Liu J., Brutkiewicz R.R., Smith L.J., Hara H., Cooper D.K, & Ekser B. (2021). Genetic engineering of porcine endothelial cell lines for evaluation of human-to-pig xenoreactive immune responses. Scientific Reports, 11, 13131.

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