Cell line authentication was performed by Microsynth AG. Profiling of the human cell lines was done using highly polymorphic short tandem repeat loci (STRs). STR loci were amplified using the PowerPlex 16 HS System (Promega). Fragment analysis was done on an ABI3730xl (Life Technologies) and the resulting data were analyzed with GeneMarker HID software (Softgenetics). A549, Calu-6, and H460 cell lines matched 100% to the DNA profile of A549 (ATCC CRM-CCL-185TM; RRID:CVCL_0023 ), Calu-6 (ATCC HTB-56TM; RRID:CVCL_0236 ), and NCI-H460 [H460] (ATCC HTB-177TM; RRID:CVCL_0459 ), respectively. SW 1573 cell line matched 93,3% to the DNA profile of the SW 1573 [SW-1573, SW1573] (ATCC CRL-2170TM; RRID:CVCL_1720 ).
Genemarker hid software
Manufactured by SoftGenetics
Sourced in United States
GeneMarker HID is a software application designed for DNA analysis. It provides tools for visualizing and analyzing genetic data from various laboratory instruments, enabling researchers to identify and interpret genetic profiles.
Automatically generated - may contain errors
Lab products found in correlation
Powerplex 16 hs system, by Promega (5 mentions)
Abi 3730xl, by Thermo Fisher Scientific (5 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Venor gem onestep kit, by Minerva Biolabs (1 mentions)
Hepes, by Thermo Fisher Scientific (1 mentions)
5 protocols using genemarker hid software
1
Cell Line Authentication via STR Profiling
2
Cell Line Characterization and Validation
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All cell lines were a kind gift from Dr. Johannes Czernin’s group (University of California Los Angeles). Cells were cultured in Rosewell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin solution at 37 °C and 5% CO2. Cells were routinely assessed for mycoplasma contamination using the Venor®GeM OneStep kit (Minerva Biolabs, Berlin, Germany). All used cell lines underwent polymorphic short tandem repeat loci (STRs) profiling to rule out cross-contaminations (Microsynth, Balgach, Switzerland). STR loci were amplified using the PowerPlex® 16 HS System (Promega, Walldrof, Germany). Fragment analysis was done on an ABI3730xl (Life Technologies, Carlsbad, CA, USA) and the resulting data were analyzed with GeneMarker HID software (Softgenetics, State College, PA, USA).
3
Cell Line Authentication Using STR Profiling
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Pellet of 1 × 106 cells was washed twice using PBS, dissolved in 0.5 mL 90% ethanol, and shipped to Microsynth AG (Balgach, Switzerland) for cell authentication. Profiling of human cell lines A549, H1975, and H1650 was completed using highly polymorphic short tandem repeat (STR) loci. STR loci were amplified using the PowerPlex 16 HS System (Promega, Madison, WI, USA). Fragment analysis was completed on an ABI3730xl (Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA) and the resulting data were analyzed with GeneMarker HID software (Softgenetics, State Collage, PA, USA).
4
Generation and Characterization of B4GALNT2-Expressing Cell Lines
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LS174T(ATCCR® Number: CL-188™) cell line was transfected with an expression vector for the short form of B4GALNT2 cDNA cloned in pcDNA3 or with the empty vector as detailed previously [8 (link)], generating the two B4GALNT2-expressing clones S2 and S11 and the polyclonal negative control Neo population, respectively. Cells were cultured in DMEM supplemented with 10% FBS and antibiotics in a humidified incubator with a 5% CO2 atmosphere at 37 °C. B4GALNT2 enzymatic activity was measured as the difference between the incorporation of radioactive GalNAc on fetuin and asialofetuin, as previously described [5 (link)]. B4GALNT2 mRNA was measured by real-time (RT) PCR as previously described [17 (link)]. Western blot analysis with an anti-Sda antibody KM694 and an anti-sLex antibody (CSLEX1) was performed as detailed elsewhere [8 (link)]. Cell lines were genotyped using highly polymorphic short tandem repeat loci, which were amplified using the PowerPlex® 16 HS System (Promega). Fragment analysis was done on an ABI3730xl (Life Technologies), and the resulting data were analyzed with GeneMarker HID software (Softgenetics) by Microsynth (Switzerland). Reports are available on request.
5
Characterization of PGRMC1 Mutants in Breast Cancer Cells
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MCF7 and T47D cells were obtained from ATCC (Manassas, Virginia) and authenticated by Microsynth AG (Balgach, Switzerland) on October 7, 2016. Profiling of human cell lines was done using highly-polymorphic short tandem repeat loci (STRs). STR loci were amplified using the PowerPlex® 16 HS System (Promega, Madison, Wisconsin). Fragment analysis was done on an ABI3730xl (Thermo Fisher Scientific, Waltham, Massachusetts) and the resulting data were analyzed using GeneMarker HID software (Softgenetics, State College, Pennsylvania).
Cells were stably transfected with expression plasmid pcDNA3.1 containing hemagglutinin (HA)-tagged PGRMC1 wild-type or HA-tagged PGRMC1 mutants S57A, S181A and S57A/S181A, as described elsewhere [35 (link)]. MCF7 and T47D cells were maintained in RPMI 1640 medium (Thermo Fisher Scientific, Waltham, Massachusetts), supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, Massachusetts), 100 units/ml penicillin/streptomycin (Thermo Fisher Scientific, Waltham, Massachusetts) and 25 mM HEPES (Thermo Fisher Scientific, Waltham, Massachusetts) in a humidified incubator at 37°C with 5% CO2.
Cells were stably transfected with expression plasmid pcDNA3.1 containing hemagglutinin (HA)-tagged PGRMC1 wild-type or HA-tagged PGRMC1 mutants S57A, S181A and S57A/S181A, as described elsewhere [35 (link)]. MCF7 and T47D cells were maintained in RPMI 1640 medium (Thermo Fisher Scientific, Waltham, Massachusetts), supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, Massachusetts), 100 units/ml penicillin/streptomycin (Thermo Fisher Scientific, Waltham, Massachusetts) and 25 mM HEPES (Thermo Fisher Scientific, Waltham, Massachusetts) in a humidified incubator at 37°C with 5% CO2.
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