Cells were cultured at 39.5 °C in D-MEM/F-12 medium (Gibco) supplemented with 10% fetal bovine serum, 2% chicken serum (Sigma), penicillin/streptomycin mix, and 10 μM 2-mercaptoethanol (Gibco) in the presence or absence of 500 μM auxin. To plot growth curves, each cell line was cultured in three different wells of 24 well-plates and passaged every 12 h or 24 h. Cell number was determined by flow cytometry using plastic microbeads (07313-5; Polysciences). Cell solutions were mixed with the plastic microbeads suspension at a ratio of 10:1, and viable cells determined by forward scatter and side scatter were counted when a given number of microbeads was detected by flow cytometry.
Plastic microbeads
Manufactured by Polysciences
Plastic microbeads are small, spherical particles made from plastic materials. They are typically used in various laboratory applications, such as in filtration, extraction, or as model systems for studying particle behavior. The core function of plastic microbeads is to provide a controlled and uniform medium for these laboratory procedures.
Automatically generated - may contain errors
Lab products found in correlation
Chicken serum, by Merck Group (4 mentions)
2 mercaptoethanol, by Thermo Fisher Scientific (4 mentions)
Dmem f12 medium, by Thermo Fisher Scientific (4 mentions)
Penicillin streptomycin mix, by Thermo Fisher Scientific (2 mentions)
Pcr4 topo, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
4 protocols using plastic microbeads
1
Cell Culture Growth Curves with Auxin
2
Inducible FOXP1A Expression in Myocytes
3
Characterizing DNA Repair Mechanisms in Cell Lines
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Cells were cultured at 39.5 °C in D-MEM/F-12 medium (Gibco) supplemented with 10% fetal bovine serum, 2% chicken serum (Sigma), Penicillin/Streptomycin mix, and 10 μM 2-mercaptoethanol (Gibco) in the presence or absence of 1 μg/ml Dox. The cell lines used in this study are shown in Table 1 . To plot growth curves, each cell line was cultured in three different wells of 24 well-plates and passaged every 12 h. Cell number was determined by flow cytometry using plastic microbeads (07313-5; Polysciences). Cell solutions were mixed with the plastic microbead suspension at a ratio of 10:1, and viable cells determined by forward scatter and side scatter were counted when a given number of microbeads were detected by flow cytometry. mCherry positive cells were detected by FL2-H as shown in Fig. 2 A.
Cell lines used in this study.
| Genotype | Selective marker | Reference |
|---|---|---|
| WT CL18 | [16] (link) | |
| WT + DR-GFP + I-SceI (Tet-On) | Puro/Neo | This study |
| WT + DR-GFP + I-SceI and BRC4 (Tet-On) | Puro/Neo/Bsr | This study |
| WT + BRC4 (Tet-On) | Neo | This study |
| WT + BRC4 A1504S (Tet-On) | Bsr | This study |
| WT + BRC4 (Tet-On) + hsRAD51 | Neo/Bsr | This study |
| BRCA2+/− | Bsr/Puro | [15] (link) |
| BRCA2+/− + BRC4 (Tet-On) | Bsr/Puro/Neo | This study |
| XRCC4/− + DR-GFP + I-SceI (Tet-On) | Neo/Puro/His | This study |
| XRCC4/− + BRC4 (Tet-On) | Puro/Neo/His | This study |
| XRCC3−/− | Bsr/His | [17] (link) |
| RAD51−/− + hsRAD51 | Puro/Bsr/Neo/Hyg | [18] (link) |
4
Inducible FOXP1A Expression in Myocytes
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Full length, murine FOXP1A (long isoform) (NM_001012505.1) was cloned by
amplified pcr into pCR4-TOPO (Invitrogen, Carlsbad, CA) and inserted under an
inducible suCMV promoter into the lentiviral expression vector,
EF1a-TetR(GFP-Bsd; Cat#: LVP1172; GenTarget Inc, San Diego CA.). After packaging
as described above, ~90% confluent C2C12 myocytes were infected in DMEM
media containing 10X Polybrene. Infections were carried out at a dose of
~100 virus particles per cell for 18 h prior to serum withdrawal-mediated
differentiation (Supplementary
Figure 5 ). A GFP-Blasticidin (Fluorescent-Antibiotic) Fusion dual
marker under the RSV promoter allows doxycycline induction of green
fluorescence, realtime monitoring of lentivirus’ expression.
C2C12 myocytes were cultured at 39.5°C in D-MEM/F-12 medium
(Gibco) supplemented with 10% fetal bovine serum, 2% chicken serum (Sigma),
penicillin/streptomycin mix, and 10 μM 2-mercaptoethanol (Gibco) in the
presence or absence of 1 μg/ml Dox. Growth curves were determined by flow
cytometry of C2C12 cells attached to plastic microbeads (07313-5; Polysciences).
At various times post-differentiation, cells were fixed for immunofluorescence
or harvested for Western blotting and RT-qPCR.
amplified pcr into pCR4-TOPO (Invitrogen, Carlsbad, CA) and inserted under an
inducible suCMV promoter into the lentiviral expression vector,
EF1a-TetR(GFP-Bsd; Cat#: LVP1172; GenTarget Inc, San Diego CA.). After packaging
as described above, ~90% confluent C2C12 myocytes were infected in DMEM
media containing 10X Polybrene. Infections were carried out at a dose of
~100 virus particles per cell for 18 h prior to serum withdrawal-mediated
differentiation (
Figure 5
marker under the RSV promoter allows doxycycline induction of green
fluorescence, realtime monitoring of lentivirus’ expression.
C2C12 myocytes were cultured at 39.5°C in D-MEM/F-12 medium
(Gibco) supplemented with 10% fetal bovine serum, 2% chicken serum (Sigma),
penicillin/streptomycin mix, and 10 μM 2-mercaptoethanol (Gibco) in the
presence or absence of 1 μg/ml Dox. Growth curves were determined by flow
cytometry of C2C12 cells attached to plastic microbeads (07313-5; Polysciences).
At various times post-differentiation, cells were fixed for immunofluorescence
or harvested for Western blotting and RT-qPCR.
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