Flow Cytometry Analysis of THP-1 Cells

THP-1 cells were plated in 24-well tissue culture treated plates (Costar) as described above. Cells were removed from the well using TrypLE (Invitrogen) incubation for 10 min at 37°C. Cells were collected on ice and all subsequent steps were performed at 4°C. Cells were pelleted and resuspended in FACS buffer (0.1% azide, 2% fetal calf serum in PBS) containing 11 μg/mL IgG (Jackson). Cells were then stained with PerCP/Cy5.5 anti-mouse/human CD11b antibody (Biolegend, #101227) and eFluor 450 anti-human CD14 antibody (eBioscience, CD14 eFlour 450) before fixation in 2% (w/v) paraformaldehyde (PFA) and analyzed using flow cytometry. Analysis was done using FlowJo software (TreeStar, Inc. vX.0.7 and v9.5.2).

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Starr T., Bauler T.J., Malik-Kale P, & Steele-Mortimer O. (2018). The phorbol 12-myristate-13-acetate differentiation protocol is critical to the interaction of THP-1 macrophages with Salmonella Typhimurium. PLoS ONE, 13(3), e0193601.

Publication 2018

24-well tissue culture treated plates TrypLE

Anti antibody Azide Buffer Cd11b Cells Cy5 5 Fetal calf serum Flow cytometry Human Mouse Paraformaldehyde Thp 1 cells Tissue

Corresponding Organization : National Institutes of Health

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Protocol cited in 15 other protocols

Variable analysis

independent variables

Incubation of THP-1 cells with TrypLE for 10 min at 37°C to remove cells from the well

dependent variables

Expression of CD11b and CD14 on THP-1 cells, as analyzed by flow cytometry

control variables

Tissue culture treated plates (Costar) used to plate THP-1 cells
All subsequent steps performed at 4°C after cell collection
Cells resuspended in FACS buffer containing 11 μg/mL IgG (Jackson) before staining
Cells fixed in 2% (w/v) paraformaldehyde (PFA) before analysis

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

Based on most similar protocols

Incubate THP-1 cells with Trypsin-EDTA for 5 min to detach the cells from the culture plate before staining (Protocol 3).

Apply FcR blocking reagent to cells to decrease non-specific antibody binding before staining (Protocol 3).

Stain the cells with a panel of fluorescently-labeled antibodies targeting specific cell surface markers (e.g., CD14, CD11b, CD86, CD209, HLA-DR, CD83, CD11c, CD40, CD80, HLA-ABC, CD63, CD81) to assess the phenotype of THP-1 cells or differentiated macrophages (Protocols 3 and 5).

Perform flow cytometry analysis using an Accuri C6 flow cytometer (Protocol 3) or FACS Aria III (Protocol 5) to quantify the expression of cell surface markers.

Analyze the flow cytometry data using FlowJo software to determine the mean fluorescence intensity (MFI) of the stained cell populations (Protocols 1, 3, and 5).

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