Human iPSCs or derived‐EBs were dissociated to into a single‐cell suspension by TrypLE (Life Technologies, Carlsbad, CA), and washed with a buffer for FACS (PBS plus 1% FBS and 1 mM EDTA). The cells were resuspended in the FACS buffer, and labeled with fluorochrome‐conjugated anti‐human CD34‐APC (Miltenyi Biotec, #130‐090‐954), CD45‐PE (Miltenyi Biotec, #130‐080‐201), CD45‐ Brilliant Violet 605 (BioLegend, San Diego, CA, #304042), CD235a‐Pacific Blue (BioLegend, #349108) antibodies. Isotype‐matched control antibodies were used to determine the background staining. The erythrocyte enucleation was evaluated by DRAQ5 staining (Thermo Scientific, #62251, 1:2,000 dilution). To characterize human iPSCs, the primary antibodies anti‐human TRA‐1‐60 (Millipore, Billerica, MA, #MAB4360) and SSEA‐4 (Millipore, #MAB4303) were used. Their recognitions were detected by respective secondary antibodies anti‐mouse IgM‐Alexa Fluor 555 (Thermo Scientific, #A21426) and anti‐mouse IgG‐Alexa Fluor 555 (Thermo Scientific, #A21422). Flow cytometric analysis was performed on a FACSCalibur or LSR II analyzer (BD Biosciences). Data analysis was performed using FlowJo or FCS Express software.
Anti mouse igm alexa fluor 555
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Anti‐mouse IgM‐Alexa Fluor 555 is a fluorescently labeled antibody that recognizes mouse immunoglobulin M (IgM). It is designed for use in various immunoassay and cell-based applications that require the detection of mouse IgM.
Automatically generated - may contain errors
Lab products found in correlation
Tryple, by Thermo Fisher Scientific (2 mentions)
Anti human tra 1 60, by Merck Group (2 mentions)
Alexa fluor 488 anti mouse igg1, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 350 anti mouse igg2b, by Thermo Fisher Scientific (2 mentions)
Ba f8, by Developmental Studies Hybridoma Bank (2 mentions)
Sc 71, by Developmental Studies Hybridoma Bank (2 mentions)
Facscalibur, by BD (1 mentions)
Ssea 4, by Merck Group (1 mentions)
Lsrii analyzer, by BD (1 mentions)
Draq5, by Thermo Fisher Scientific (1 mentions)
Anti mouse igg alexa fluor 555, by Thermo Fisher Scientific (1 mentions)
Cd45 pe, by Miltenyi Biotec (1 mentions)
Cd45 brilliant violet 605, by BioLegend (1 mentions)
Anti human cd34 apc, by Miltenyi Biotec (1 mentions)
Lrs fortessa, by BD (1 mentions)
Sh800s cell sorter, by Sony (1 mentions)
Lc3b 2775, by Cell Signaling Technology (1 mentions)
Anti ubiquitin p4d1, by Cell Signaling Technology (1 mentions)
4 protocols using anti mouse igm alexa fluor 555
1
Multiparametric Flow Cytometry for Characterizing Human iPSCs and Derived Cell Types
2
Reprogramming and Differentiation of iPSCs
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To evaluate the successful reprogramming of PBMCs into iPSCs, cells were dissociated into single-cell suspensions with TrypLE TM (Life Technologies). They were then washed and resuspended in PBS with 1% BSA (wt/v). They were labeled with the primary antibody anti-human TRA-1-60 (Millipore). For the subsequent detection, iPSCs were labeled with secondary anti-mouse IgM-Alexa®Fluor555 (Thermo Scientific) antibody.
To compare iPSCs and the 5-HT-organoids they were differentiated into, iPSCs and 5-HT-organoids were dissociated with TrypLE TM and Gentle cell dissociation reagent (STEM cells technologies) respectively. Cells were washed and resuspended in PBS with 1% BSA (wt/v), following which they were fixed and permeabilized with Cytofix/Cytoperm solution (BD Biosciencence) according to manufacturer's instructions. Samples were subsequently labeled using AlexaFluor®488 conjugated anti-human TUJ1 and AlexaFluor®647 conjugated antihuman TPH2 antibodies (ThermoScientific). The former is a general neuronal marker, whereas the latter is specific for serotonergic neurons.
All samples were analyzed on a BD LRS Fortessa (BD Biosciences) or on a SH800S cell sorter (SONY Biotechnology). The data was processed using FlowJo TM v10.8.1 software. A full list of the antibodies used for flow cytometry and ICC is available in Table S2 in the SI.
To compare iPSCs and the 5-HT-organoids they were differentiated into, iPSCs and 5-HT-organoids were dissociated with TrypLE TM and Gentle cell dissociation reagent (STEM cells technologies) respectively. Cells were washed and resuspended in PBS with 1% BSA (wt/v), following which they were fixed and permeabilized with Cytofix/Cytoperm solution (BD Biosciencence) according to manufacturer's instructions. Samples were subsequently labeled using AlexaFluor®488 conjugated anti-human TUJ1 and AlexaFluor®647 conjugated antihuman TPH2 antibodies (ThermoScientific). The former is a general neuronal marker, whereas the latter is specific for serotonergic neurons.
All samples were analyzed on a BD LRS Fortessa (BD Biosciences) or on a SH800S cell sorter (SONY Biotechnology). The data was processed using FlowJo TM v10.8.1 software. A full list of the antibodies used for flow cytometry and ICC is available in Table S2 in the SI.
3
Comprehensive Antibody Catalogue for Muscle and Calcium Signaling
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Primary antibodies against PLN (2D12), phosphorylated-NFATc1 (PA5-38301), total NFATc1 (MA3-024), SERCA2a (MA3-919), ryanodine receptor 1 (RyR, MA3-925), dihydropyridine receptor α1 subunit (DHPR, MA3-920), and calsequesterin I and II (CSQ, MA3-913) were obtained from Pierce Antibodies. The primary antibody directed against SLN was generated by Lampire Biological Laboratories (PA, USA) [28 (link)]. LC3B (2775) and p62 (GP62-C) antibodies were obtained from Cell Signaling Technology (MA, USA) and Progen Biotechnik (Heidelberg, Germany), respectively. The primary antibody against actin (A2066) and stabilin-2 (orb158499) were obtained from Sigma Aldrich (MO, USA), and Biorbyt (CA, USA), respectively. SERCA1a antibody (A52) was a kind gift from Dr. David MacLennan (University of Toronto) [29 (link)]. The primary antibodies against MHCI (BA-F8), MHCIIa (SC-71), and MHCIIb (BF-F3) were obtained from Developmental Studies Hybridoma Bank (IA, USA). Secondary antibodies for western blotting, goat anti-mouse IgG (peroxidase conjugated) and goat anti-rabbit IgG (peroxidase conjugated) were obtained from Santa Cruz Biotechnology (TX, USA). Secondary antibodies for immunofluorescence staining, Alexa Fluor 350 anti-mouse IgG2b, Alexa Fluor 488 anti-mouse IgG1, and Alexa Fluor 555 anti-mouse IgM, were obtained from Molecular Probes (OR, USA).
4
Muscle Protein Expression Analysis
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Primary antibodies against SERCA2a (2A7-A1), PLN (2D12), dynamin 2 (PA5-19800) and RyR (MA3-925) were obtained from Pierce Antibodies. The primary antibody for SERCA1a (A52) was a kind gift from Dr David MacLennan (University of Toronto) (Zubrzycka-Gaarn et al., 1984 (link)). The primary antibody directed against SLN was generated by Lampire Biological Laboratories (Fajardo et al., 2013 (link)). Anti-ubiquitin (P4D1) and anti-nitrotyrosine (189542) antibodies were obtained from Cell Signaling Technology and Cayman Chemicals, respectively. The primary antibody against α-actin (A4700) was obtained from Sigma-Aldrich. The primary antibodies against MHCI (BA-F8), MHCIIa (SC-71), MHCIIb (BF-F3), embryonic MHC (BF-F6) (Schiaffino et al., 1989 (link); Lucas et al., 2000 (link)) and dystrophin (3B7) (Nguyen thi et al., 1990 (link)) were obtained from Developmental Studies Hybridoma Bank. Secondary antibodies for western blotting, goat anti-mouse IgG (peroxidase conjugated) and goat anti-rabbit IgG (peroxidase conjugated) were obtained from Santa Cruz Biotechnology. Secondary antibodies for immunofluorescence staining, Alexa Fluor 350 anti-mouse IgG2b, Alexa Fluor 488 anti-mouse IgG1 and Alexa Fluor 555 anti-mouse IgM, were obtained from Molecular Probes.
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