FACS analysis was performed to analyze immune cell populations of innate immunity and adaptive immunity in inflammatory skin. Briefly, skin tissues were digested with collagenase D (V900893, Sigma) and Dnase 1 (D8071, Solarbio, Beijing, China) to prepare a single cell suspension. The cell suspension was then stained with zombie violet viability dye, and blocked non-specific Fc-mediated interactions with CD16/CD32 Monoclonal Antibody. Then, cells were incubated with the indicated antibody cocktail mix (Panel A or B, listed in Table 1 ) for 1 h at 4 °C with shaken every 10 min gently. Finally, the cells were resuspended in stabilizing fixative buffer (#338036, BD biosciences, San Jose, CA, USA). FACS analysis was performed using the Thermo Attune NxT machine (Waltham, MA, USA) and further analyzed using FlowJo V10 software.
Stabilizing fixative buffer
Manufactured by BD
Sourced in United States
Stabilizing fixative buffer is a laboratory reagent used to preserve and stabilize biological samples. It helps maintain the structural and chemical integrity of cells, tissues, or other biological materials during storage and processing. The buffer's core function is to prevent degradation and alterations of the sample's components.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using stabilizing fixative buffer
1
Immune Cell Profiling of Inflammatory Skin
2
Flow Cytometric Characterization of Tumor-Infiltrating T Cells
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Tumor cell suspensions were treated for 5 min on ice with ammonium-chloride-potassium lysis (ACK) buffer (Invitrogen, CA), then tumor cells (5 × 105 to 1 × 106) were stained for 20 min in the dark at 4°C with surface marker monoclonal antibodies: rat anti-mouse CD45-PE, hamster anti-mouse CD3-V450, rat anti-mouse CD4-Alexa 700, and rat anti-mouse CD8-APC-Cy7. All antibodies were purchased from BD Biosciences. Cells were subsequently fixed in stabilizing fixative buffer (BD Biosciences) and stored at 4°C in the dark until flow cytometric analysis (performed within 24 hr). Data was collected using an LSRFortessa flow cytometer (BD Biosciences) configured to detect 12 fluorochromes, and analysis was performed using FlowJo software (version 10.0; TreeStar). Initial gating used forward scatter (FSC-A) versus SSC-A plot to remove cell debris. Leukocytes were selected with CD45+ cells in CD45 versus FSC-A plot. T cells were selected by gating on CD3+ cells in CD3 versus SSC-A plot; those T cells were further selected CD8+ T cells by gating on CD4-CD8+ T cells and selected CD4+ T cells by gating on CD4+CD8− T cells.
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