pe conjugated anti human tra 1 85  (R&D Systems)

Journal: Stem cell research

Article Title: Generation of six human induced pluripotent stem cell sublines (MZT01E, MZT01F, MZT01N and MZT02D, MZT02G and MZT02H) for reproductive science research

doi: 10.1016/j.scr.2021.102204

Figure Lengend Snippet: All hiPSC sublines display equivalent features of self-renewal and pluripotency.

Article Snippet: Fibroblasts were passaged using 0.05% Tryspin (Gibco) and re-plated, the derived cells were termed MZT01 and MZT02 based on the order the biopsy was received ( and ). table ft1 table-wrap mode="anchored" t5 caption a7 Classification Test Result Data Morphology Immunofluorescence Normal panel C Phenotype Immunofluorescence Positive for self-renewal markers: Oct4, Nanog, SSEA-4, Tra-1–81 panel C Flow cytometry MZT04-D: panel B Tra 1–81: 85.9%, SSEA-4: 98.7% MZT04-J: Tra 1–81: 92.7%, SSEA-4: 81.5% MZT04-C: Tra 1–81: 84.3%, SSEA-4: 81.5% Genotype Karyotype (G-banding) and resolution 46,XX panel A Identity Microsatellite PCR (mPCR) OR STR analysis Performed Supplementary Fig. 2 16 sites tested, all three lines match each other, and the HDF line they were derived from Supplementary Fig. 2 Mutation analysis (IF APPLICABLE) Sequencing N/A Southern Blot OR WGS N/A Microbiology and virology Mycoplasma Mycoplasma testing by Luminescence Supplementary Fig. 1 Differentiation potential Pluritest Pluripotent panel E Donor screening (OPTIONAL) N/A Genotype additional info (OPTIONAL) N/A N/A Open in a separate window Characterization and validation. table ft1 table-wrap mode="anchored" t5 caption a7 Antibodies used for immunocytochemistry/flow-citometry Antibody Dilution Company Cat # and RRID Self-renewal markers goat-anti-human Oct4 1:100 Santa Cruz, sc8628 RRID: AB_653551 Self-renewal markers goat-anti-human NANOG 1:40 R&D Systems, AF1997 RRID: AB 355097 Self-renewal markers mouse-anti-human SSEA-4 1:100 Developmental Studies Hybridoma Bank, MC-813–70 RRID: AB_528477 Self-renewal markers mouse-anti-human TRA-1–81 1:100 eBiosciences, 14–8883–82 RRID: AB_891614 Pluripotency markers SSEA-4-Allophycocyanin 1:30 R&D Systems, FAB1435A RRID: AB_494994 Pluripotency markers TRA-1–85-Phycoerythrin 1:60 R&D Systems, FAB3195P RRID: AB_2066683 Pluripotency markers TRA-1–81, Alexa Fluor 488 1:60 Stemcell Technologies, 60065AD RRID: AB_2721032 Pluripotency markers Dapi 1:100 BioVision, B1098–25 RRID: AB_2336790 Secondary antibodies AF488-conjugated donkey-anti-goat 1:200 JacksonImmunoResearch, 705–546–147 RRID: AB_2340430 Secondary antibodies AF488-conjugated donkey-anti-mouse 1:200 Life Technologies, A-21131 RRID: AB_2535771 Primers Target Forward/Reverse primer (5′–3′) Reprogramming virus SeV GGA TCA CTA GGT GAT ATC GAG C/ ACC AGA CAA GAG TTT AAG AGA TAT GTA TC Reprogramming virus: KOS ATG CAC CGC TAC GAC GTG AGC GC/ ACC TTG ACA ATC CTG ATG TGG Reprogramming virus: Klf4 TTC CTG CAT GCC AGA GGA GCC C/ AAT GTA TCG AAG GTG CTC AA Reprogramming virus: c-Myc TAA CTG ACT AGC AGG CTT GTC G/ TCC ACA TAC AGT CCT GGA TGA TGA TG Open in a separate window Reagents details.

Techniques:

Journal: Stem cell research

Article Title: Generation of six human induced pluripotent stem cell sublines (MZT01E, MZT01F, MZT01N and MZT02D, MZT02G and MZT02H) for reproductive science research

doi: 10.1016/j.scr.2021.102204

Figure Lengend Snippet: Reagents details.

Article Snippet: Fibroblasts were passaged using 0.05% Tryspin (Gibco) and re-plated, the derived cells were termed MZT01 and MZT02 based on the order the biopsy was received ( and ). table ft1 table-wrap mode="anchored" t5 caption a7 Classification Test Result Data Morphology Immunofluorescence Normal panel C Phenotype Immunofluorescence Positive for self-renewal markers: Oct4, Nanog, SSEA-4, Tra-1–81 panel C Flow cytometry MZT04-D: panel B Tra 1–81: 85.9%, SSEA-4: 98.7% MZT04-J: Tra 1–81: 92.7%, SSEA-4: 81.5% MZT04-C: Tra 1–81: 84.3%, SSEA-4: 81.5% Genotype Karyotype (G-banding) and resolution 46,XX panel A Identity Microsatellite PCR (mPCR) OR STR analysis Performed Supplementary Fig. 2 16 sites tested, all three lines match each other, and the HDF line they were derived from Supplementary Fig. 2 Mutation analysis (IF APPLICABLE) Sequencing N/A Southern Blot OR WGS N/A Microbiology and virology Mycoplasma Mycoplasma testing by Luminescence Supplementary Fig. 1 Differentiation potential Pluritest Pluripotent panel E Donor screening (OPTIONAL) N/A Genotype additional info (OPTIONAL) N/A N/A Open in a separate window Characterization and validation. table ft1 table-wrap mode="anchored" t5 caption a7 Antibodies used for immunocytochemistry/flow-citometry Antibody Dilution Company Cat # and RRID Self-renewal markers goat-anti-human Oct4 1:100 Santa Cruz, sc8628 RRID: AB_653551 Self-renewal markers goat-anti-human NANOG 1:40 R&D Systems, AF1997 RRID: AB 355097 Self-renewal markers mouse-anti-human SSEA-4 1:100 Developmental Studies Hybridoma Bank, MC-813–70 RRID: AB_528477 Self-renewal markers mouse-anti-human TRA-1–81 1:100 eBiosciences, 14–8883–82 RRID: AB_891614 Pluripotency markers SSEA-4-Allophycocyanin 1:30 R&D Systems, FAB1435A RRID: AB_494994 Pluripotency markers TRA-1–85-Phycoerythrin 1:60 R&D Systems, FAB3195P RRID: AB_2066683 Pluripotency markers TRA-1–81, Alexa Fluor 488 1:60 Stemcell Technologies, 60065AD RRID: AB_2721032 Pluripotency markers Dapi 1:100 BioVision, B1098–25 RRID: AB_2336790 Secondary antibodies AF488-conjugated donkey-anti-goat 1:200 JacksonImmunoResearch, 705–546–147 RRID: AB_2340430 Secondary antibodies AF488-conjugated donkey-anti-mouse 1:200 Life Technologies, A-21131 RRID: AB_2535771 Primers Target Forward/Reverse primer (5′–3′) Reprogramming virus SeV GGA TCA CTA GGT GAT ATC GAG C/ ACC AGA CAA GAG TTT AAG AGA TAT GTA TC Reprogramming virus: KOS ATG CAC CGC TAC GAC GTG AGC GC/ ACC TTG ACA ATC CTG ATG TGG Reprogramming virus: Klf4 TTC CTG CAT GCC AGA GGA GCC C/ AAT GTA TCG AAG GTG CTC AA Reprogramming virus: c-Myc TAA CTG ACT AGC AGG CTT GTC G/ TCC ACA TAC AGT CCT GGA TGA TGA TG Open in a separate window Reagents details.

Techniques: Immunocytochemistry, Virus