Using the Neon transfection system pre-set program #16 (1400V, 20ms, 3 pulses) plasmids used for reprogramming (available from Addgene) were EN2L, ET2K, EM2K, and MIR302, along with EBNA RNA. We transfected 0.5ug of each plasmid into 4 X 10^6 cells per 10 cm Vitronectin-coated dish in DMEM/20%FBS/1X NEAA. After 4-5 days, cells were maintained on vitronectin (5ug/cm^2) in rhesus iPSC media: Essential 8 Flex Medium (Thermofisher) supplemented with 100ug/L rhNodal (R&D #3218-ND), 1.94ug/ml glutathione, 1% Chemically Defined Lipid Concentrate (ThermoFisher), 1% GlutaMAX (ThermoFisher) and 1x Pen/Strep Amphotericin B (Lonza, 17-745E). This line was provided by the Thomson lab and assayed for standard pluripotency markers and had a normal karyotype (supplemental figure 1). rhIPSC89 and rhiPSC90 were maintained on MEFs (50K/cm^2) (Thermofisher, A34180) in hESC media: 80% DMEM/F12, 20% KOSR, 10ng/ml bFGF, 1mM L-glutamine, 0.1mM B-Me, 1% NEAA and 1x Pen/Strep Amphotericin B (Lonza, 17-745E). For feeder-free culture, healthy iPSC colonies were manually picked and plated on reduced growth factor Matrigel plates (50ug/cm^2) in MEF conditioned hESC media supplemented with bFGF prior to use. Human Embryonic stem cell line H9 (WiCell) and CRX +/tdtomato were maintained in reduced growth factor Matrigel (50ug/cm^2) in mTeSR1 plus with 1x Pen/Strep Amphotericin B (Lonza, 17-745E).
Pen strep amphotericin b
Manufactured by Lonza
Pen/Strep Amphotericin B is a sterile, liquid media supplement that contains penicillin, streptomycin, and amphotericin B. It is used to prevent bacterial and fungal contamination in cell culture applications.
Automatically generated - may contain errors
Lab products found in correlation
Glutamax, by Thermo Fisher Scientific (1 mentions)
Chemically defined lipid concentrate, by Thermo Fisher Scientific (1 mentions)
Essential 8 flex medium, by Thermo Fisher Scientific (1 mentions)
Rhnodal, by R&D Systems (1 mentions)
L glutamine, by Corning (1 mentions)
Primocin, by InvivoGen (1 mentions)
Amphotericin b, by Lonza (1 mentions)
Dulbecco s modified eagle medium, by Lonza (1 mentions)
2 protocols using pen strep amphotericin b
1
Rhesus iPSC Generation and Maintenance
2
Isolation and Culture of Lung Fibroblasts from IPF Patients
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Lung fibroblasts were derived from surgical lung biopsies (SLBs) from patients who exhibited slowly or rapidly progressing IPF (slow IPF and rapid IPF, respectively) over the first year after diagnosis12 (link). Primary normal lung fibroblasts were derived from non-fibrotic lung samples lacking any evidence of disease. Histologically-proven IPF and normal lung samples were dissociated mechanically as previously described in detail13 (link). Briefly, SLBs were mechanically dispersed in 150 cm3 flasks (Corning), combined with Dulbecco’s modified Eagle Medium (Lonza), containing 15% fetal bovine serum (Cell Generation), 0.1% Pen/Strep/Amphotericin B (10,000 U of penicillin/ml, 10,000 μg of streptomycin/ml, and 25 μg Amphotericin B/ml) (Lonza), 0.1% (29.20 mg/mL) L-glutamine (Corning), and Primocin at 100 μg/ml (Invivogen). Isolated fibroblasts were incubated at 37 °C in 10% CO2 until confluency was reached. The cells were passaged 3 to 4 times before use in the experiments described herein. In the present study, we analyzed a total of 4 normal fibroblast lines, 7 fibroblast lines derived from IPF patients who showed slow progression, and 7 fibroblast lines derived from IPF patients who showed rapid progression.
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