Vero E6 cells were dissociated, fixed with 4% paraformaldehyde at room temperature for a minimum of 24 h, washed once with PBS and permeabilized with 1× perm-wash buffer (BD Biosciences) for 5 min. SARS-CoV nucleocapsid (N) antibody (clone 1C7C7) (kindly provided by Thomas Moran, Icahn School of Medicine at Mount Sinai, New York, NY) conjugated to AlexaFluor 647 was diluted 1:400 in perm-wash buffer, and added directly to samples. Samples were then incubated at room temperature for 40 min in the dark. After staining, samples were washed once with 1× perm-wash buffer, once with PBS, resuspended in FACS buffer (PBS supplemented with 1% FBS), and acquired on a Gallios flow cytometer (Beckman-Coulter). For all viral infections, analysis was performed with FlowJo software (v10.7.1, Becton Dickinson), excluding cell doublets and debris and gating according to mock-infected populations.
Perm wash buffer
Manufactured by Beckman Coulter
Sourced in United States
Perm-wash buffer is a laboratory reagent used to maintain the permeability and structural integrity of cells during sample preparation. It is designed to facilitate the entry of antibodies or other detection agents into the cells for intracellular staining and analysis. The buffer composition helps to preserve the cellular morphology and antigen presentation without compromising the analytical process.
Automatically generated - may contain errors
Lab products found in correlation
Perm wash buffer, by BD (3 mentions)
Gallios flow cytometer, by Beckman Coulter (2 mentions)
Annexin 5, by BD (1 mentions)
Fc blocker, by BD (1 mentions)
Cytoflex, by Beckman Coulter (1 mentions)
Fix perm buffer, by R&D Systems (1 mentions)
Perm wash buffer, by R&D Systems (1 mentions)
F4 80, by BD (1 mentions)
Pd l1, by BD (1 mentions)
Cytoflex cytometer, by Beckman Coulter (1 mentions)
Cytofix cytoperm solution, by BD (1 mentions)
4 protocols using perm wash buffer
1
SARS-CoV-2 Nucleocapsid Protein Detection
2
Multicolor Flow Cytometry for Immune Profiling
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For cell surface staining, cells were washed with phosphate-buffed saline (PBS) and blocked with Fc blocker (BD Biosciences, San Jose, California, USA). Fluorochrome-labeled antibodies (Annexin-V, CD45, CD11c, CD4, CD8, CD11b, Ly6G, Ly6C, PD-1, PD-L1, F4/80, CD56, CD86, HLA-DR, CD206, and CD44) were obtained from BD Biosciences (Franklin Lakes, USA), added, and stained for 30 min as described.54 (link) For intracellular staining, cells were permeabilized with Fix/Perm buffer (#FC009, R&D Systems, Minneapolis, Minnesota, USA) for 20 min and then washed with Perm/Wash buffer (R&D Systems). Fluorochrome-labeled antibodies (IFNγ and TNFα) (#562019, #561062, BD Biosciences) were diluted in Perm/Wash buffer and stained for 30 min as described.55 (link) All samples were analyzed on a CytoFlex flow cytometer (Beckman Coulter, California, USA).
3
SARS-CoV-2 Nucleocapsid Protein Detection
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Vero E6 cells were dissociated, fixed with 4% paraformaldehyde at room temperature for a minimum of 24 h, washed once with PBS and permeabilized with 1× perm-wash buffer (BD Biosciences) for 5 min. SARS-CoV nucleocapsid (N) antibody (clone 1C7C7) (kindly provided by Thomas Moran, Icahn School of Medicine at Mount Sinai, New York, NY) conjugated to AlexaFluor 647 was diluted 1:400 in perm-wash buffer, and added directly to samples. Samples were then incubated at room temperature for 40 min in the dark. After staining, samples were washed once with 1× perm-wash buffer, once with PBS, resuspended in FACS buffer (PBS supplemented with 1% FBS), and acquired on a Gallios flow cytometer (Beckman-Coulter). For all viral infections, analysis was performed with FlowJo software (v10.7.1, Becton Dickinson), excluding cell doublets and debris and gating according to mock-infected populations.
4
Erythroid Differentiation Analysis by Flow Cytometry
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Differentiated CD34+ cells collected at day 7 of erythroid differentiation were washed with PBS. For flow cytometry analysis, non-specific binding of antibodies was blocked with 10% FBS with human IgG (20 μg/mL) at 4°C for 20 min. Following blocking, the surface markers of the cells were stained with an antibody cocktail containing CD235a-PE/Cy7, and CD71-APC at 4°C for 30 min. The cells were washed with PBS, re-suspended in 1x Cytofix/Cytoperm solution (BD Biosciences), and fixed for 20 min at 4°C. Following fixation, cells were washed with Perm/Wash Buffer (BD Biosciences) and stained with anti-HbF-PE for 30 min at room temperature. For isotype control, IgG-PE was used in place of HbF-PE. Finally, the cells were washed twice with Perm/Wash Buffer, resuspended in PBS containing 2% FBS, and then analyzed by flow cytometry using a Beckman Coulter CytoFlex cytometer. Both percentages of various cell populations as well as events (cells) per μL were collected. The HbF+ cells were found within the CD71+ bright cell population. HU was used as a positive control throughout the experiment.
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