Rat spermatogonial stem cell lines were cultured on mitomycin-C treated MEFs in spermatogonial culture medium (SG medium) as described [69 (link)]. The cells were passaged at 1:3 dilutions onto a fresh monolayer of MEFs every 10–14 days at 3 × 104 cells/cm2. For passaging, cultures were first harvested by gently pipetting them free from the MEFs. After harvesting, the clusters of spermatogonia were dissociated by gentle trituration with 20–30 strokes through a p1000 pipette in their SG culture medium. The dissociated cells were pelleted at 200× g for 4 min, and the number of cells recovered during each passage was determined by counting them on a hemocytometer. Feeder Removal Micro Beads (Miltenyi Biotec, Bergisch-Gladbach, Germany) were used to deplete MEFs while co-culturing with rat spermatogonial stem cells for gene expression and protein studies. The protocol for co-culturing the rat spermatogonial stem cells with STO cells was performed as in [56 (link)]. The separation of STO cells from rat spermatogonial stem cells was carried out using magnetic-activated cell sorting (MACS) from (Miltenyi Biotec, Bergisch-Gladbach, Germany).
Feeder removal microbeads
Manufactured by Miltenyi Biotec
Sourced in Germany
Feeder removal microbeads are magnetic beads designed to facilitate the separation and removal of feeder cells from cell cultures. They provide a simple and efficient way to isolate the desired cell population for further applications.
Automatically generated - may contain errors
Lab products found in correlation
Cd146 microbeads, by Miltenyi Biotec (2 mentions)
Opti mem, by Thermo Fisher Scientific (2 mentions)
Mgso4, by Carl Roth (1 mentions)
Sodium chloride (nacl), by Carl Roth (1 mentions)
Potassium chloride (kcl), by Carl Roth (1 mentions)
Nah2po4, by Merck Group (1 mentions)
Liberase th, by Roche (1 mentions)
Hiseq 2500 system, by Illumina (1 mentions)
Nebnext ultra rna library prep kit, by New England Biolabs (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Magnetic cell sorter, by Miltenyi Biotec (1 mentions)
Wnt10b, by R&D Systems (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Collagenase dispase solution, by Roche (1 mentions)
8 protocols using feeder removal microbeads
1
Rat Spermatogonial Stem Cell Culture
2
Isolation and Culture of Cardiac Cell Types
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For culture, cardiac fibroblasts were isolated from 6 to 8 week-old Gata4flox/flox, Gata6flox/flox, and Gata4flox/floxGata6flox/flox mice as previously described [34 (link)]. Whole hearts without atria were quickly cut into small pieces in ice-cold PBS. Digestion was performed using Liberase TH (Roche) with SADO buffer mix solution (20 mM HEPES–NaOH (Roth, Nr. 9105, pH 7.6), 130 mM NaCl (Roth, Nr. 9265), 3 mM KCl (Roth, Nr. 6781), 1 mM NaH2PO4 (Sigma, Nr. S5011), 4 mM Glucose (Roth, Nr. HN06), 3 mM MgSO4 (Roth, Nr. 2278) in sterile, filtered dH20). The digested cell solution was plated and after 3 h washed once with PBS and changed to DMEM supplemented with 10% FCS. Isolated fibroblasts were infected with AdCre or Adβ-Gal for 3 h and after two washing steps with PBS cultured in DMEM supplemented with 2% FCS for 48 h. Cells were passaged two times at maximum before harvesting.
For immediate RNA or protein-isolation, cardiac fibroblasts and cardiac endothelial cells were isolated from hearts of adult mice using CD146 microbeads and feeder removal microbeads with MACS (magnetic cell separation) technology from Miltenyi Biotec.
Adult ventricular cardiomyocytes were isolated as previously described using a Langendorff system [24 (link)].
For immediate RNA or protein-isolation, cardiac fibroblasts and cardiac endothelial cells were isolated from hearts of adult mice using CD146 microbeads and feeder removal microbeads with MACS (magnetic cell separation) technology from Miltenyi Biotec.
Adult ventricular cardiomyocytes were isolated as previously described using a Langendorff system [24 (link)].
3
RNA-seq Analysis of Pluripotent Stem Cells
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RNA was isolated from samples harvested from cPP and PPd15 cultures using an RNeasy mini kit (QIAGEN, cat. no. 74104). Feeder removal microbeads (Miltenyi Biotec, 130-095-531) were used to deplete cPP cells of 3T3 feeders prior to RNA extraction. All RNA samples had an RNA integrity number >9. RNA-seq libraries were generated using the NEBNext Ultra RNA Library Prep Kit (NEB, E7530L) and sequenced on an Illumina HiSeq 2500 system generating single-end reads of 100 bp. Table S1 contains metadata for these and public datasets used for the RNA-seq gene expression analysis. Full details of how RNA-seq reads were aligned and analyzed can be found in the Supplemental Experimental Procedures .
4
Primary Esophageal Cell Culture Protocol
5
Modeling HBV Replication in iPS-HPCs
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Replication of HBV can be mimicked by transfection of plasmids expressing the HBV genome into hepatic host cells32 (link). Briefly, iPS-HPCs were enriched from feeder mouse embryonic fibroblasts using feeder removal microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) and a magnetic cell sorter (Miltenyi Biotec), and seeded onto collagen-coated dishes. Host iPS-HPCs were transfected with 1 μg plasmids expressing the HBV genome, D-IND60, in 100 μL Opti-MEM (Thermo Fisher Scientific) using ViaFect transfection reagent complex (Promega), and were cultured for 3 days. Then, 1 μM LMV, 1 μM TFV, and 1,000 IU/ml IFN-α were added to the medium for drug inhibition assays. For IFN-α inhibition assays, 1,000 IU/ml IFN-α with or without 0.5–2.0 μM JAKi (Pyridone 6) was added to the medium.
6
Mouse iPS Cell Aggregation and EB Formation
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Mouse iPS cells were harvested by dissociation using 0.25% trypsin-EDTA (Invitrogen) with feeder cells. Then, dissociated cells were separated into iPS cells and feeder cells using Feeder Removal MicroBeads (MACS; Miltenyi Biotec). For EB culture, the iPS cells were reaggregated using Nunclon Sphera round-bottom 96-well plates (Thermo Fisher Scientific Inc.) in a culture medium (3000 cells/100 μl per well). Fresh medium was added to the culture on day 3, and Wnt10b (500 ng/ml; R&D Systems) was added on day 6.
7
Cardiac cell isolation and transcriptome analysis in TAC mouse model
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Transverse aortic constriction (TAC) or sham surgery was performed in eight-week-old male and female Fgf23fl/fl/cre− and Fgf23fl/fl/cre+ mice by subjecting the aorta to a defined constriction as previously described (Grund et al., 2019a (link)). In brief, a 7–0 silk suture was tied around the aortic arch between the branches of the brachiocephalic trunk and the left common carotid artery and a blunted 27-gauge needle, after which the needle was removed to create a defined constriction. The procedure for sham-operated animals was identical, but the aorta was not ligated. Two weeks after surgery, mice were sacrificed as described above.
Cardiac specific cell types were isolated from mice after sham and TAC as previously described (Appari et al., 2017 (link)). In brief, ventricular cardiac myocytes were isolated using a Langendorff system, cardiac endothelial cells and fibroblasts were isolated using MACS technology with CD146 microbeads followed by feeder removal microbeads (Miltenyi Biotec). All isolated cell types were directly used for RNA extraction according to standard protocol followed by deep-sequencing analysis as previously described (Grund et al., 2019b (link)).
Cardiac specific cell types were isolated from mice after sham and TAC as previously described (Appari et al., 2017 (link)). In brief, ventricular cardiac myocytes were isolated using a Langendorff system, cardiac endothelial cells and fibroblasts were isolated using MACS technology with CD146 microbeads followed by feeder removal microbeads (Miltenyi Biotec). All isolated cell types were directly used for RNA extraction according to standard protocol followed by deep-sequencing analysis as previously described (Grund et al., 2019b (link)).
8
Isolation and Culture of Murine Satellite Cells
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