Unspecific binding sites were blocked with phosphate-buffered saline supplemented with 1% fetal bovine serum and either Fc blocker (CD16/32, eBioscience) or whole mouse IgG (Sigma Aldrich) before cells were incubated with the respective monoclonal antibody at 4°C for 30 min. After incubation, cells were washed twice with phosphate-buffered saline and analyzed by flow cytometry with a BD FACSCalibur, BD FACS CantoII, BD LSRII or BD Fortessa (BD Biosciences, NJ, USA). Data were subsequently analyzed using FlowJo software (Oregon, USA). A complete list of antibodies used is given in the Online Supplementary Appendix.
Whole mouse igg
Manufactured by Merck Group
Sourced in Macao
Whole mouse IgG is a purified immunoglobulin G (IgG) fraction isolated from mouse serum. IgG is the most abundant antibody isotype found in the body and plays a crucial role in the adaptive immune response.
Automatically generated - may contain errors
Lab products found in correlation
Protein g dynabead, by Thermo Fisher Scientific (3 mentions)
Lamelli buffer, by Bio-Rad (3 mentions)
Flag m2, by Merck Group (3 mentions)
Fortessa, by BD (2 mentions)
Ip lysis buffer, by Bio-Rad (2 mentions)
Fc blocker cd16 32, by Thermo Fisher Scientific (1 mentions)
Lsr 2, by BD (1 mentions)
Facscalibur, by BD (1 mentions)
Facscanto 2, by BD (1 mentions)
Ebioscience foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (1 mentions)
Brilliant stain buffer, by BD (1 mentions)
5 protocols using whole mouse igg
1
Flow Cytometry Cell Staining Protocol
2
GATA6 Protein Co-Immunoprecipitation
3
GATA6 Knockout Differentiation Protocol
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GATA6-/- cells were differentiated to day 2 of the Duncan lab differentiation protocol. After 24 h of differentiation, the cells were cultured in the absence or presence of doxycycline. Samples were collected at day 2. Cells were lysed with IP lysis buffer (ThermoFisher/Pierce, IL, #87787) containing 1x HALT proteinase inhibitor cocktail (ThermoFisher/Pierce, IL, #78443) and Universal Nuclease (ThermoFisher/Pierce, IL, #88701). Lysates were pre-cleared with Protein G Dynabeads (ThermoFisher Scientific, NY, #10004D) for 2 h at 4°C with shaking. The precleared samples were split equally and incubated with either 1 μg of FLAG M2 (Sigma-Aldrich, MO, #F1804) or 1 μg whole mouse IgG (Sigma-Aldrich, MO, #12-371) antibodies overnight at 4°C. Protein G Dynabeads were then added and the mixture was incubated for 2 h at 4°C. IP lysis buffer was used to wash the beads five times, before elution in 1x Lamelli buffer (BioRad, CA, #1610747) at 95°C for 10 min before analysis by western blot.
4
Multiparametric Flow Cytometry of Cells
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Dead cells were stained using LIVEDEAD (Thermo) for 15 min on ice in PBS. For traditional flow cytometry, nonspecific antibody binding was blocked by incubation with an anti-CD16/32 antibody (clone 2.4G2; UCSF Ab production facility) and whole mouse IgG (Sigma) on ice for 15 min, and the cells were stained with fluorophore-conjugated antibodies on ice for 20 min. Cells were kept on ice and analyzed on a BD Fortessa (BD Biosciences). For spectral flow, cells were incubated with anti-CD16/32 APC-Fire750 and anti-CD64 PECy7 for 15 min on ice before the addition of the rest of the fluorophore-conjugated antibodies were added on ice for 20 min in Brilliant Stain Buffer (BD Biosciences). For GATA6 staining, cells were fixed and permeabilized with the eBioscience™ Foxp3 / Transcription Factor Staining Buffer Set (Thermo) for 30 minutes followed by staining with GATA6-PE (Cell Signaling Technologies) at 1:400 for 30 minutes on ice. Cells were kept on ice until analyzed on a 5 laser Cytek Aurora (Cytek Biosciences). Data were analyzed in FlowJo (FlowJo, LLC). For UMAP analysis, equivalent numbers of cells in each were concatenated and all markers were included except LiveDead and GFP (for the retroviral transductants).
5
GATA6 Protein Co-Immunoprecipitation
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GATA6Ex4Δ1/Δ2:ind GATA6 cells were differentiated to mesendoderm (day 2). Between days 1 and 2, the cells were cultured in the absence or presence of 10ng/ml doxycycline to induce GATA6–3xFLAG expression at endogenous levels. At Day 2, cells were washed once with PBS and lysed with IP lysis buffer (ThermoFisher/Pierce, IL, #87787) with HALT proteinase inhibitor cocktail (ThermoFisher/Pierce, IL, #78443) and Universal Nuclease (ThermoFisher/Pierce, IL, #88701). The sample was incubated at room temperature for 10 minutes before centrifugation to remove cell debris. The lysate was pre-cleared with pre-washed Protein G Dynabeads (ThermoFisher Scientific, NY, #10004D) for 2 hours at 4°C. An input sample was taken and the remaining lysate split equally and incubated with either 1 μg of FLAG M2 (Sigma-Aldrich, MO, #F1804) or 1 μg whole mouse IgG (Sigma-Aldrich, MO, #12–371) antibodies overnight at 4°C. The following day, pre-washed Protein G Dynabeads were added and the mixture was incubated for 2 hours at 4°C. The beads were then washed 5 times with IP lysis buffer and eluted in 1x Lamelli buffer (BioRad, CA, #1610747) at 95°C for 10 minutes. Samples were then analyzed by western blot. Antibodies used for co-IP listed in Table S8 .
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