Megakaryocytic cell lines (HEL, Dami, CMK) were maintained in a serum‐free medium (Panserin 401, PAN‐Biotech, Aidenbach, Germany). To induce ID, the cells were grown either in Panserin 401S (PAN‐Biotech; Dami), a customized iron‐ and transferrin‐free medium based on Panserin 401, or in Panserin 401S supplemented with Panserin 401 to 1.25% (volume/volume; HEL and CMK).
Panserin 401
Manufactured by PAN Biotech
Sourced in Germany
Panserin 401 is a cell culture media product designed for the cultivation of a wide range of cell types. It provides the necessary nutrients and growth factors to support the in vitro growth and maintenance of cells. The core function of Panserin 401 is to facilitate the optimal growth and proliferation of cells in a controlled laboratory environment.
Automatically generated - may contain errors
Lab products found in correlation
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (4 mentions)
Insulin, by Harvard Bioscience (2 mentions)
Glucose, by B. Braun (2 mentions)
Penicillin, by Harvard Bioscience (2 mentions)
Ca2 mg2 free pbs, by Thermo Fisher Scientific (2 mentions)
Hepes buffer solution, by Thermo Fisher Scientific (2 mentions)
N2 supplement, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (2 mentions)
Tris buffer, by Merck Group (1 mentions)
Cb 150 e3, by Binder (1 mentions)
Ultraculturetm, by Lonza (1 mentions)
Purecol, by Advanced BioMatrix (1 mentions)
Dmem f12, by Merck Group (1 mentions)
Red blood cell lysis buffer, by Merck Group (1 mentions)
Cd45r b220, by BD (1 mentions)
Macs column, by Miltenyi Biotec (1 mentions)
Cd11b, by BD (1 mentions)
Ter119, by BD (1 mentions)
40 μm cell strainer, by BD (1 mentions)
11 protocols using panserin 401
1
Inducing Iron Deficiency in Megakaryocytes
2
Cultivating Stratified Cells for Vascular Research
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One or two SGE were placed onto the abluminal side of the adventitia and the luminal side of the intima and media. The three different dEAC layers with SGE were cultivated for 5 days in a medium consisting of Panserin 401 (PAN-Biotech, Aidenbach, Germany) supplemented with insulin (8.7 µg/mL; Biochrom, Berlin, Germany), penicillin (30 U/mL; Biochrom), glucose (0.15%; B. Braun Melsungen, Melsungen, Germany), phosphate-buffered saline (PBS; Ca2+/Mg2+-free PBS; 0.172 mg/mL; Gibco by Life Technologies, Carlsbad, CA, USA), HEPES buffer solution (23.43 µM; Invitrogen, Carlsbad, CA, USA) and N-2 supplement (0.1 µL/mL; Invitrogen) in an incubator (CB 150 E3; Binder, Tuttlingen, Germany) at 37°C, 5% CO2 and humidity of 95%. The serum-free medium was conditioned with 10% fetal calf serum (FCS). At the end of the experiments, dEAC layers with SGE were fixed with a 4% paraformaldehyde (PFA) solution per well for 1 h at 4°C and washed two times using hydroxymethyl-aminomethane (TRIS) buffer (Merck) and stored at 4°C.22 (link) The experiments were repeated in triplicate with SGE from different animals for each of the layers in one-well plate.
3
Serum-Free Adaptation of MDA-MB-231 Cells
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MDA-MB-231 cells (DSMZ, DSMZ no. ACC-732) were maintained in the basal medium Dulbecco’s Modified Eagle`s Medium/Nutrient Mixture F-12 Ham (DMEM/F-12; Sigma-Aldrich) supplemented with 10% fetal calf serum (FCS; Sigma-Aldrich) at 37 °C in a humidified incubator at 5% CO2. Cells were subcultured after reaching an optical confluence of 70–90%. Cells were adapted to the serum-free growth media UltraCULTURETM (UC; Lonza) and PanSerin 401 (PS; PAN-Biotech) by lowering the content of the original serum-containing medium in a stepwise fashion. The adaptation was performed in five subcultivation steps, replacing each time 20% serum-containing base medium by UC or PS. Cell culture flasks were coated with bovine collagen type I (PureCol, Advanced BioMatrix).
4
Isolation and Culture of Murine Erythroid Progenitors
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All animal experiments were approved by the governmental review committee on animal care of the state Baden-Württemberg, Germany (reference number DKFZ215). At E13.5 Balb/c mouse embryos were dissected from the uteri of female mice euthanized by CO2 inhalation. Fetal livers were resuspended in PBS/0.3% BSA and passed through a 40-μm cell strainer (BD Biosciences). Fetal liver cells (FLCs) were treated with 9 ml Red Blood Cell Lysis Buffer (Sigma-Aldrich) to remove erythrocytes. For sorting Ter119− erythroid progenitors, FLCs were incubated with rat antibodies against the following surface markers: GR1, CD41, CD11b, CD14, CD45R/B220, CD4, CD8 and Ter119 (BD Pharmingen), and 42.2.2 for 30 min at 4°C. After washing, cells were incubated for 30 min at 4°C with anti-rat antibody-coupled magnetic beads and negatively sorted with MACS columns according to the manufacturer's instructions (Miltenyi Biotech). Sorted CFU-E cells were cultivated for 12–14 hours in Panserin 401 (PAN-biotech) supplemented with 50 μM β-mercaptoethanol and 0.5 U/ml EPO alfa. Before the experiments, cells were growth factor-depleted for 60 min.
5
Cultivation of CD4+ T-cell Lines
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The CD4+ T-cell lines Jurkat (from acute lymphoblastic leukemia) [43 (link)], Molt-4 [44 (link)], MT-2 (HTLV-1 in vitro transformed) [45 (link)], Tesi (Tax-1-transformed) [46 (link)], the Jurkat-derived cell line SVT35 (carrying an NF-κB-driven CD14 reporter gene) [47 (link)], and the HTLV-2-transformed T-cell lines MoT [48 (link)] and C3-44-Mo [49 (link)] were cultured in RPMI 1640M with L-glutamine (0.35 g/L), penicillin/streptomycin, and 20% (Tesi) or 10% (all other cell lines) fetal calf serum. For cultivation of Jurkat, SVT35, Molt-4, and Tesi, media were supplemented with 45% Panserin 401 (PAN-Biotech, Aidenbach, Germany), and Tesi cells were additionally cultured in media supplemented with 40 U/mL interleukin-2 (Roche Diagnostics GmbH, Mannheim, Germany).
6
Isolation and Culture of Spiral Ganglion Cells
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SGCs, including the SGN, were harvested from early postnatal (2–5 days) Sprague Dawley rats. The tissue preparation was performed in accordance with the German “Law on protecting animals” and with the Directive 86/609/EEC of the European Communities Council for protection of animals used for experimental purposes. Preparation and dissection of the spiral ganglia were previously described in detail.17 (link) In brief, animals were decapitated, spiral ganglia prepared, collected, and the cells subsequently dissociated. Cell number was determined by Trypan blue exclusion assay in a Neubauer chamber. A cell number of 20 × 104 SGC/mL was adjusted in SGC-medium. This SGC-medium based on Panserin 401 (Pan-Biotech) supplemented with insulin (8.7 µg/mL; Biochrom Ltd), penicillin (30 U/mL; Biochrom Ltd), glucose (0.15%; B. Braun Melsungen AG), PBS (Ca2+/Mg2+-free PBS; 0.172 mg/mL; Gibco by Life Technologies), HEPES-buffer solution (23.43 µM; Invit-rogen), and N2-supplement (0.1 µL/mL; Invitrogen).
7
Cell Culture Protocols for T-cell and B-cell Lines
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The CD4+ T-cell line Jurkat (from acute lymphoblastic leukemia; ATCC, LGC Standards GmbH, Wesel, Germany) was cultured in RPMI 1640 (GIBCO, Life Technologies, Darmstadt, Germany) supplemented with 45% Panserin 401 (PAN-Biotech, Aidenbach, Germany), 10% fetal calf serum (FCS), L-glutamine (0.35 g/l) and penicillin/streptomycin (0.12 g/l each). The HTLV-1 in vitro transformed CD4+ T-cell line MT-2 [72 (link)] (kindly provided by Ralph Grassmann, deceased, FAU, Erlangen, Germany) was cultured in RPMI 1640 containing 10% FCS, L-glutamine and penicillin/streptomycin. 293T cells (kindly provided by Ralph Grassmann, deceased, FAU, Erlangen, Germany) were cultured in DMEM (GIBCO, Life Technologies), 10% FCS, L-glutamine and penicillin/streptomycin. The human Epstein-Barr virus (EBV)-positive B-cell line Raji containing the CD4 surface receptor (Raji/CD4+) was a kind gift from Vineet N. Kewal Ramani (NIH, Frederick,Maryland, USA) and was cultured in RPMI 1640M, Panserin, 10% FCS, L-glutamine and Pen/Strep containing 500 μg/ml geneticin [73 (link)].
8
Expansion of Human T Cell Blasts
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Whole blood samples were collected from the patient and control donors. PBMCs were isolated by Ficoll-Paque density gradient (Lymphoprep; Proteogenix) from blood samples using standard procedures. Expansion of T cell blasts were obtained by incubating PBMCs for 72 h with 2.5 µg ml−1 PHA (Sigma-Aldrich) in Panserin 401 (Pan Biotech) supplemented with 5% human male AB serum (BioWest), 100 U ml−1 penicillin, and 100 µg ml−1 streptomycin. After 3 d, dead cells were removed by Ficoll-Paque density gradient, and blasts were maintained in culture with 100 or 1,000 UI ml−1 IL-2.
9
Culturing CD4+ Jurkat and 293T Cells
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CD4+ Jurkat T-cells (from acute lymphoblastic leukemia) were cultured in RPMI 1640 with L -glutamine (0.35 g/l; GIBCO, Life Technologies, Darmstadt, Germany) supplemented with 45% Panserin 401 (PAN-Biotech, Aidenbach, Germany), 10% fetal calf serum (FCS; Sigma-Aldrich, Darmstadt, Germany) and penicillin/streptomycin (0.12 g/l each; Sigma-Aldrich). 293T cells were cultured in DMEM (GIBCO, Life Technologies), 10% FCS, L -glutamine and penicillin/streptomycin.
10
Cell Culture Conditions and Stimulation Assays
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HEK293T cells were cultured in DMEM (Gibco) supplemented with 10% FBS (Invitrogen). EBV-B cells were cultured in RPMI (Gibco) supplemented with 10% FBS. HVS-T cells were cultured in a 1:1 mixture (by volume) of RPMI and Panserin 401 (PAN Biotech) supplemented with 10% FBS, GlutaMAX (350 µg/ml; Gibco), gentamicin (0.1 mg/ml; Gibco), and rhIL-2 (20 IU/ml; Roche).
The cells were starved for 2 h by incubation in serum-free RPMI. The cells were then left unstimulated or were stimulated with rhIFN-α 2b (105 IU/ml; Schering), rhIL-23 (100 ng/ml; R&D Systems), rhIL-12 (20 ng/ml; R&D Systems), or rhIL-10 (50 ng/ml; PeproTech) for 5 min to assess the phosphorylation of JAKs, and for 30 min to assess the phosphorylation of STATs. For quantitative RT-PCR, cells were stimulated for 6 h with IL-10 (50 ng/ml) or IFN-α (105 IU/ml). For RNA-seq experiments, cells were starved for 1.5 h in RPMI containing 1% FCS and were stimulated for 2 h with IFN-α (105 IU/ml) and IL-21 (100 ng/ml). RNA was extracted with the Zymoresearch kit. For scRNA-seq experiments, PBMCs were either left unstimulated or were stimulated with 100 ng/ml of IL-23 or 103 IU/ml IFN-α2b for 6 h.
The cells were starved for 2 h by incubation in serum-free RPMI. The cells were then left unstimulated or were stimulated with rhIFN-α 2b (105 IU/ml; Schering), rhIL-23 (100 ng/ml; R&D Systems), rhIL-12 (20 ng/ml; R&D Systems), or rhIL-10 (50 ng/ml; PeproTech) for 5 min to assess the phosphorylation of JAKs, and for 30 min to assess the phosphorylation of STATs. For quantitative RT-PCR, cells were stimulated for 6 h with IL-10 (50 ng/ml) or IFN-α (105 IU/ml). For RNA-seq experiments, cells were starved for 1.5 h in RPMI containing 1% FCS and were stimulated for 2 h with IFN-α (105 IU/ml) and IL-21 (100 ng/ml). RNA was extracted with the Zymoresearch kit. For scRNA-seq experiments, PBMCs were either left unstimulated or were stimulated with 100 ng/ml of IL-23 or 103 IU/ml IFN-α2b for 6 h.
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