Single-cell suspensions of BC cell lines were obtained from cell cultures after trypsinization, fixed with Fixation Medium A (Invitrogen, CA, USA) for 15 min at RT and permeabilized with 0.2% Triton X-100 (Wako, Osaka, Japan) for 30 min at RT. Cells were stained with 1 μg/mL primary antibody to ERα (anti-ERα mouse mAb (1D5), Abcam, Tokyo, Japan) at RT for 20 min [30 (link)]. Afterward, flow cytometry was performed on a BD Aria II Flow Cytometer system (BD Biosciences, CA, USA) using a quantitative kit (QIFIKIT, Dako, CA, USA), which was used for the quantitative determination of cell antigen in accordance with the manufacturer’s instructions. Two washes were performed at each step with ice-cold Hanks’ Balanced Salt Solution (HBSS, Gibco, Life Technologies, CA, USA) containing 2% FBS. SP1 was used for flow cytometry to quantify IHC-PIDs and determine the amount protein, but the quantitative kit used for flow cytometry could only recognize the mouse antibody, thus 1D5 was used in this study.
Fixation medium a
Manufactured by Thermo Fisher Scientific
Sourced in United States
Fixation Medium A is a liquid solution designed for the preservation and stabilization of biological samples. It is used to maintain the structural integrity of cells, tissues, and other biological materials prior to further analysis or processing.
Automatically generated - may contain errors
Lab products found in correlation
Permeabilization medium b, by Thermo Fisher Scientific (2 mentions)
Triton x 100, by Fujifilm (1 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (1 mentions)
Mitotracker deep red, by Thermo Fisher Scientific (1 mentions)
Er id red, by Enzo Life Sciences (1 mentions)
Mito id red, by Enzo Life Sciences (1 mentions)
Golgi id green, by Enzo Life Sciences (1 mentions)
Accuri 6 flow cytometry, by BD (1 mentions)
Golgiplug protein transport inhibitor, by BD (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Tnf αpe cy7 mab11, by BD (1 mentions)
Fortessa flow cytometer, by BD (1 mentions)
Ifn γ fitc 4s b3, by BD (1 mentions)
Cd3 alexa fluor 700, by BD (1 mentions)
Cd56 pe cy7, by BD (1 mentions)
Ifn γ apc, by BD (1 mentions)
Cd16 apc cy7, by BD (1 mentions)
Cd107a pe cy5, by BD (1 mentions)
Golgistop, by BD (1 mentions)
6 protocols using fixation medium a
1
Quantification of Estrogen Receptor-Alpha
2
Multiparametric Analysis of Protein and Organelle Dynamics
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For HC and LC protein, cells (1 x 10 6 ) were fixed using a 1:1 solution of Fixation Medium A (Invitrogen) and flow cytometry buffer (PBS with 5% BSA) for 15 min at RT and then incubated with Permeabilisation Medium B (Invitrogen) containing 1:20 goat f(ab')2 antihuman IgG Alexa Fluor conjugated to 488 (Invitrogen, excitation/emission: ~500/520 nm) and goat f(ab')2 anti-human kappa conjugated to APC (Biolegend, excitation/emission: ~650/675 nm) for 15 min, before being washed and analysed. HC and LC mRNA were investigated using a PrimeFlow RNA kit with custom probes (Affymetrix, Merck) following the manufacturer protocol. Quantitation of organelles was performed by staining with Golgi-ID Green (excitation/emission: ~450/530 nm), ER-ID red (excitation/emission: ~580/660 nm), Mito-ID red (excitation/emission: ~558/690 nm; all from Enzo Life Sciences) and MitoTracker Deep Red (Invitrogen, excitation/emisssion: ~644/665 nm) using the manufacturers' protocols. DAPI (excitation/emisssion: ~340/450 nm) or Sytox nuclear (excitation/emission: ~444/480) counter-stain were used for nuclear localization.
3
Intracellular Cytokine Staining Protocol
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For IFNγ and IP10 intracellular cytokine staining studies, cells were incubated with the respective ligands as indicated above. Following centrifugation, supernatants were discarded and cell pellets were resuspended in fixation medium A (100μl, ThermoFisher Scientific) and incubated in dark at RT for 15 mins. PBS-BSA-Sodium azide buffer (2 ml) was added into each tube and then washed twice. At the end of the second washing step, permeabilization medium B (50μl, ThermoFisher Scientific) containing anti-IFNγ-FITC and anti-CD4-PE cocktail were added onto cells. For IP10 staining, anti-CD14-FITC and anti-IP10-PE (S1A Table ) were added and the samples were incubated in dark for 30 mins. Cells were washed twice, resuspended in 500μl PBS-BSA-Sodium azide buffer, transferred to FACS tubes and analyzed in BD Accuri 6 Flow Cytometry.
4
Cytokine Profiling of Activated T Cells
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Transduced T cells were co-cultured with target cells at a 1:2 ratio or with 3 μg/mL PMA (Sigma Aldrich) and 1 μg/mL Ionomycin (Sigma Aldrich) for 5 h in the presence of GolgiPlug Protein Transport Inhibitor (BD Biosciences). Cells were then stained for surface markers, fixed with Fixation Medium A (Thermo Fisher Scientific), washed, and stained with IL2-APC (MQ1-17H2, BD), TNFα-PE-Cy7 (MAB11, BD), IFNγ-FITC (4S.B3, BD), MIP1β-PerCP-Cy5.5 (D21-1351, BD), and IL-10-PE/Dazzle 594 (JES3-9D7, Biolegend) antibodies in Fixation Medium B (Thermo Fisher Scientific) for 20 min. Following a final wash, cells were analyzed on a BD Fortessa flow cytometer.
5
Antibody Intracellular Expression Profiling
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Intracellular expression of the heavy and light chains of antibody molecules was determined by staining cells with fluorescently‐labeled antibodies specific for heavy or light chains (Roy, Miro‐Quesada, et al., 2017 ). Briefly, the cells were centrifuged, washed with FACS buffer and fixed with Fixation Medium A (Life Technologies) for 15 min at room temperature. Next, the cells were washed with FACS buffer and stained for 15 min at room temperature with the staining solution comprised of goat anti‐human IgG (Fc)‐Alexa Fluor(AF)488 (Life Technologies) and goat anti‐human κ‐APC (Biolegend) in Permeabilization Medium B (Life Technologies). The stained cells were washed and resuspended in FACS buffer before analysis on an LSRII for the APC and AF488 double‐positive population. Data analysis was performed using FlowJo software.
6
Phenotyping NK Cell Activation
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Enzyme-linked immunosorbent assay plates were coated with measles antigen and incubated with samples diluted 1:20, or PBS as negative control, for 2 hours at 37°C. Natural killer cells were isolated from buffy coats collected from blood bank donors. CD107a-PE/Cy5 (BD), brefeldin A (Sigma), GolgiStop (BD), and NK cells were added to the antigen-coated plate and incubated for 5 hours at 37°C. Next, cells were transferred to a V-bottom plate and stained with CD56-PE/Cy7, CD16-APC/Cy7, and CD3-AlexaFluor700 (all BD) for 15 minutes at room temperature. Cells were washed and fixed in Fixation Medium A (Life Technologies) before intracellular staining with MIP-1β-PE and IFN-γ-APC (BD) in Permeabilization Medium B (Life Technologies) for 15 minutes at room temperature. The percentages of positive NK cells for CD107a, IFN-γ, and MIP-1β were determined by flow cytometry.
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