BV2 cells were treated with 0.01% (v/v) FITC-conjugated latex beads (Sigma, L1030) and 0.05% fetal bovine serum in DMEM at 37 °C for 4 h. For flow cytometry, BV2 cell pellets were collected by centrifugation at 300 g for 5 min and then resuspended in BD Cytofix™ Fixation Buffer (BD Biosciences, 554655). Subsequently, the cells were incubated for 10 min at 37 °C and ultimately resuspended in PBS. Flow cytometry was performed on a CytoFLEX flow cytometer (Beckman), collecting 30,000 cells per sample, and data was analyzed using FlowJo. Specifically, each sample file was loaded, and single-cell populations were gated based on forward scatter and side scatter properties to exclude debris and doublets. FITC+ cell populations were identified based on univariate histograms. The percentage of microglia that phagocytosed beads (% Phagocytosis) was calculated by dividing the number of FITC+ cells by the total number of gated cells.
Fitc conjugated latex beads
Manufactured by Merck Group
Sourced in Germany
FITC-conjugated latex beads are fluorescent particles made from polystyrene latex and conjugated with the fluorescent dye fluorescein isothiocyanate (FITC). They are used as a reference standard or a tracer in various analytical and research applications.
Automatically generated - may contain errors
Lab products found in correlation
Cytofix fixation buffer, by BD (2 mentions)
Cytoflex flow cytometer, by Beckman Coulter (2 mentions)
Facsaria 3, by BD (1 mentions)
Dm6000b, by Leica (1 mentions)
μ slide angiogenesis, by Ibidi (1 mentions)
Cytofix cytoperm, by BD (1 mentions)
Matrigel, by BD (1 mentions)
Vascular endothelial growth factor (vegf), by Thermo Fisher Scientific (1 mentions)
Cxcl12, by Thermo Fisher Scientific (1 mentions)
Hydroethidine, by Thermo Fisher Scientific (1 mentions)
Vectashield with dapi, by Vector Laboratories (1 mentions)
Rhodamine phalloidin, by Biotium (1 mentions)
Anti α tubulin fitc, by Merck Group (1 mentions)
4 protocols using fitc conjugated latex beads
1
Phagocytosis Quantification in BV2 Cells
2
Phagocytosis in Pregnant Murine Macrophages
3
Phenotypic and Functional Analysis of Bone Marrow-Derived Macrophages
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Single-donor BMCs (107) or pooled BMCs (75×106) of three individual donors were sorted (FACSAria III; BD) after staining for CD14/CD16/CD45 and plated on 24-well plates or 4-well chamber slides in RPMI 1640 medium with 10% FCS and antibiotics. Phagocytosis of FITC-conjugated latex beads (1∶1000; Sigma-Aldrich) and hydroethidine (10 µg/ml; Invitrogen, Darmstadt, Germany) staining for spontaneous intracellular superoxide production in PBMC- or sorted BMC-derived macrophages were determined by flow cytometry after 24 hours in culture. Cytoskeletal filaments in cultured BMCs were visualized by fluorescence microscopy (Leica DM6000B, Wetzlar, Germany) after cell fixation/permeabilization (Cytofix/Cytoperm; BD) following incubation with anti-α-tubulin-FITC (Sigma-Aldrich) mAb and rhodamine-phalloidin (Biotium, Hayward, CA). Vectashield with DAPI (Vector Laboratories, Peterborough, UK) was used as mounting medium and to stain cell nuclei. In another experimental setup the sorted BMCs were cultured in angiogenesis μ-slide (IBIDI, Martinsried, Germany) on Matrigel (BD) supplemented with 50 ng/ml VEGF and 100 ng/ml Cxcl12 (both from Peprotech, Hamburg, Germany).
4
Microglia Phagocytosis Assay Protocol
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Microglial phagocytosis was detected using immunofluorescence and flow cytometry assays.
For immunofluorescence, BV2 cells (5 × 104 cells/well) were seeded in 24-well plates coated with poly-l -lysine and cultured overnight for adherence; 60 μM of daidzin was added for 48 h. Then, FITC-conjugated latex beads (0.01%, v/v; Sigma, L1030) were preincubated in DMEM containing 0.05% (v/v) FBS at 37 °C for 1 h, and were added to the cells. After incubation for 4 h, immunofluorescence analysis was conducted.
For flow cytometry, BV2 cells (1 × 105 cells/well) were seeded in six-well plates and cultured overnight. After the knockdown of Aldh2 for 48 h, oAβ40 (1 μM) was added for 24 h, followed by the addition of preincubated beads for 4 h. Cell pellets were collected by centrifugation at 500×g for 5 min. Simultaneously, an appropriate amount of BD Cytofix™ Fixation Buffer (BD Biosciences, 554655) was preheated at 37 °C for 5 min. The pellets were then resuspended in 300 μL Fixation Buffer and 300 μL PBS and incubated for 10 min at 37 °C. Subsequently, centrifugation was performed at 500×g for 5 min, and the cells were resuspended in 1 mL of PBS for flow cytometry analysis using a CytoFLEX flow cytometer (Beckman Coulter, USA). Data were analyzed and plotted using FlowJo software.
For immunofluorescence, BV2 cells (5 × 104 cells/well) were seeded in 24-well plates coated with poly-
For flow cytometry, BV2 cells (1 × 105 cells/well) were seeded in six-well plates and cultured overnight. After the knockdown of Aldh2 for 48 h, oAβ40 (1 μM) was added for 24 h, followed by the addition of preincubated beads for 4 h. Cell pellets were collected by centrifugation at 500×g for 5 min. Simultaneously, an appropriate amount of BD Cytofix™ Fixation Buffer (BD Biosciences, 554655) was preheated at 37 °C for 5 min. The pellets were then resuspended in 300 μL Fixation Buffer and 300 μL PBS and incubated for 10 min at 37 °C. Subsequently, centrifugation was performed at 500×g for 5 min, and the cells were resuspended in 1 mL of PBS for flow cytometry analysis using a CytoFLEX flow cytometer (Beckman Coulter, USA). Data were analyzed and plotted using FlowJo software.
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