60,000 microglia were plated on a NuncTM Lab-TekTM II 8 Chamber SlideTM (Thermo Fischer Scientific, #154534PK, Waltham, MA, USA) and incubated with the indicated oligoribonucleotides complexed to LyoVec (InvivoGen #LYEC-RNA, San Diego, CA, USA). After 2 h, microglia were analyzed by phagocytosis bead assay, as described [36 (link)]. Briefly, red fluorescent latex beads (size 1 μm; Sigma Aldrich, #L2778, St. Louis, MO, USA) were pre-opsonized in FCS for 1 h at 37 °C. Subsequently, the suspension was diluted at 1:5 in DMEM (Invitrogen #41965062, Carlsbad, CA, USA) and added to microglial cell cultures at 0.01% (v/v). One hour later, microglia were washed 3x with PBS and fixed with 4% paraformaldehyde (PFA). Immunolabeling with Iba1 antibody and subsequent microscopic analysis were performed, as described above. Red signal intensity within Iba1-positive image areas was quantified using FiJi software [37 (link)], as described previously [36 (link)].
Red fluorescent latex beads
Manufactured by Merck Group
Sourced in United States
Red fluorescent latex beads are spherical particles made of latex polymer material that emit red fluorescent light when exposed to the appropriate excitation light. These beads are commonly used as labeling and detection agents in various bioanalytical and imaging applications.
Automatically generated - may contain errors
Lab products found in correlation
Deltavision fluorescence microscopy system, by Cytiva (2 mentions)
Tcs sp5, by Leica (2 mentions)
Lsr 2 flow cytometer, by BD (2 mentions)
Lyec rna, by InvivoGen (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Tcs sp5 2, by Leica (1 mentions)
Fitc klh, by Merck Group (1 mentions)
Cytochalasin b, by Merck Group (1 mentions)
Zymosan, by Merck Group (1 mentions)
Red coloured zymosan, by Thermo Fisher Scientific (1 mentions)
Purified rabbit igg, by Merck Group (1 mentions)
Fv1000d, by Olympus (1 mentions)
Axioskop microscope, by Zeiss (1 mentions)
Hilyte fluor 488 labeled amyloid β peptide 25 35, by Merck Group (1 mentions)
Hilyte fluor 488 labeled amyloid β peptide 25 35, by AnaSpec (1 mentions)
Middlebrook 7h9 broth, by BD (1 mentions)
Accuri c6, by BD (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Axiophot fluorescent microscope, by Leica (1 mentions)
Tcs sp5 confocal microscope, by Leica (1 mentions)
11 protocols using red fluorescent latex beads
1
Microglial Phagocytosis Assay with Opsonized Beads
2
Quantifying Phagocytosis in Microglia
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To quantify phagocytosed beads of iMGs, optimally cultured iMGs for 21 days were treated with 2 μl red fluorescent latex beads (2 μm, Sigma-Aldrich, St. Louis, MO, USA) for 2 h at 37 °C. Phagocytic activity was then stopped by adding 2 ml ice-cold PBS. The cells were washed twice with ice-cold PBS, fixed, stained with a microglial marker (IBA-1), and counterstained with DAPI. The cells were analyzed using confocal microscopy (TCS SP5-II , Leica).
3
Quantifying Microglial Phagocytic Capacity
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To assess the phagocytic activity, NEL-MG at a density of 2 × 105 cells/mL were seeded on a 12 mm coverslip in 24-well cell culture dishes. NEL-MG were treated with 2 μL of red fluorescent latex beads (2 μm, Sigma-Aldrich, St. Louis, MO, USA), HiLyte™ Fluor 488-labeled amyloid β peptide 25–35 (2 μL), or pHrodo-conjugated synaptosomes for 2 h at 37 °C. HiLyte™ Fluor 488-labeled amyloid β peptide 25–35 (Anaspec, AS-633308) was prepared according to the manufacturer’s protocol. Phagocytic activity was then stopped by adding 2 mL of ice-cold PBS. The cells were washed twice with ice-cold PBS, fixed, stained with a microglial marker (IBA-1), and counterstained with DAPI. The cells were analyzed using confocal microscopy (TCS SP5, Leica) and a DeltaVision fluorescence microscopy system (Applied Precision).
4
IgM Modulation of Macrophage Phagocytosis
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The effect of IgMhigh and IgMlow on the antibody-mediated clearance was assessed in mouse phagocytic J774A.1 cell line. Red fluorescent latex beads (2 μm in diameter, Sigma-Aldrich) were coated with purified IgMhigh and IgMlow in a 1:7 ratio and incubated for 45 min at 37 °C. IgM promotes the clearance of small particles and apoptotic microparticles by macrophages (44 (link)). The IgM-coated latex beads were suspended in serum-free culture medium and added to J774A.1macrophages at a 5:1 ratio. After 180-min incubation, the cells were washed in PBS to remove nonengulfed beads. The cells were detached with trypsinization and fixed in 4% paraformaldehyde and subsequently analyzed with a BD LSR II flow cytometer. The macrophage population was identified as described before (70 (link)), and the proportion of macrophages containing ingested particles was determined based on the fluorescent signal of the ingested latex beads.
5
Phagocytic Uptake of Antigens by γδ T Cells
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Freshly isolated γδ T cells (4 × 104) were incubated at 28°C with FITC-conjugated keyhole limpet hemocyanin (FITC-KLH; Sigma-Aldrich), red fluorescent latex beads (1 µm; Sigma-Aldrich, L-2778) or FITC-labeled Aeromonas hydrophila (A.h) at a cell/bead ratio of 1:10. Cells in the control group for active phagocytosis were incubated on ice. After 4 h, trypan blue (200 µg/mL) was added to quench the fluorescence of KLH/beads/A.h that had not been internalized for 5 min at 4°C. In parallel, γδ T cells incubated with FITC-KLH, red fluorescent beads, and FITC-A.h (28°C for 4 h) in the presence of cytochalasin B (80 µg/mL; Sigma-Aldrich) were set as controls. Then, cells were washed thrice with PBS before FCM analysis.
6
Zymosan Phagocytosis Assay in RAW264.7 Cells
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Red-coloured zymosan (Invitrogen) or zymosan (Sigma) was opsonized with bovine serum albumin and incubated at 0.2 mg ml−1 with cells at 37 °C. The cells were either subjected to live-cell imaging or fixed after 1 h. Live images were obtained with a confocal microscope (Olympus FV1000D) at 37 °C in a 5% CO2 atmosphere.
Wild-type or GAS7-knockout RAW264.7 cells grown on cover glasses were incubated with Red-coloured zymosan (Invitrogen) or red fluorescent latex beads (2 μm, Sigma) coated with purified rabbit IgG (Sigma), for 1 h at 37 °C. The cells were washed, fixed with 4% paraformaldehyde for 20 min and mounted. Approximately 100–200 cells from six to seven randomly selected fields were examined, to determine the number of incorporated particles.
Wild-type or GAS7-knockout RAW264.7 cells grown on cover glasses were incubated with Red-coloured zymosan (Invitrogen) or red fluorescent latex beads (2 μm, Sigma) coated with purified rabbit IgG (Sigma), for 1 h at 37 °C. The cells were washed, fixed with 4% paraformaldehyde for 20 min and mounted. Approximately 100–200 cells from six to seven randomly selected fields were examined, to determine the number of incorporated particles.
7
Imaging Ependymal Cell Dynamics in Mouse Brains
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Brains of experimental mice were extracted, sliced in half to expose the ependymal of the lateral ventricles and placed in pre-warmed, pre-oxygenated artificial cerebrospinal fluid (125mM NaCl, 2.5mM KCl, 1.25mM NaH2PO4, 2mM CaCl2, 1mM MgCl2, 25mM NaHCO3, 25mM Glucose, pH 7.35). Brains were placed on a Zeiss Axioskop microscope and imaged with a 5x objective (Plan-Neofluor, 0.15NA) and a 10x objective (Fluor, 0.5NA) using a Photometrics CoolSnap HQ CCD camera at 30fps. Red fluorescent latex beads (cat# L3530-1mL, Sigma-Aldrich) were diluted 1:100 from stock and 10μL of diluted beads was added to the ventricles. Bead tracking analysis was performed using the MTrack2 plugin in FIJI.
8
Quantifying Microglial Phagocytic Activity
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To assess the phagocytic activity, NEL-MG at a density of 2 × 10 5 cells/mL were seeded on a 12-mm coverslip in 24-well cell culture dishes. NEL-MG were treated with 2 μL of red fluorescent latex beads (2 μm, Sigma-Aldrich, St. Louis, MO, USA),
HiLyte TM Fluor 488-labeled amyloid β peptide 25-35 (2 μL), or pHrodo-conjugated synaptosomes for 2 h at 37 °C. HiLyte TM Fluor 488-labeled amyloid β peptide 25-35 (Anaspec, AS-633308) was prepared according to the manufacturer's protocol.
Phagocytic activity was then stopped by adding 2 mL of ice-cold PBS. The cells were washed twice with ice-cold PBS, fixed, stained with a microglial marker (IBA-1), and counterstained with DAPI. The cells were analyzed using confocal microscopy (TCS SP5, Leica) and a DeltaVision fluorescence microscopy system (Applied Precision).
HiLyte TM Fluor 488-labeled amyloid β peptide 25-35 (2 μL), or pHrodo-conjugated synaptosomes for 2 h at 37 °C. HiLyte TM Fluor 488-labeled amyloid β peptide 25-35 (Anaspec, AS-633308) was prepared according to the manufacturer's protocol.
Phagocytic activity was then stopped by adding 2 mL of ice-cold PBS. The cells were washed twice with ice-cold PBS, fixed, stained with a microglial marker (IBA-1), and counterstained with DAPI. The cells were analyzed using confocal microscopy (TCS SP5, Leica) and a DeltaVision fluorescence microscopy system (Applied Precision).
9
IgM Regulates Phagocytic Clearance
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The effect of IgM high and IgM low on the antibody-mediated clearance was assessed in mouse phagocytic J774A.1 cell line. Red fluorescent latex beads (2 µm in diameter, Sigma-Aldrich) were coated with purified IgM high and IgM low in a 1:7 ratio and incubated for 45 min at 37˚C IgM Promotes the Clearance of Small Particles and Apoptotic Microparticles by Macrophages (Litvack et al, 2011) . The IgM-coated latex beads were suspended in serum-free culture medium and added to J774A.1macrophages at a 5:1 ratio. After 180 min incubation, cells were washed in PBS to remove non-engulfed beads. Cells were detached with trypsinization and fixed in 4% PFA, and subsequently analyzed with BD LSR II flow cytometer. The macrophage population was identified as described before (Yu et al, 2019) and the proportion of macrophages containing ingested particles was determined based on the fluorescent signal of the ingested latex beads.
10
BCG-GFP Phagocytosis and Cytokine Assay
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Mycobacterium bovis Bacille Calmette Guérin (BCG)-expressing green fluorescent protein (GFP; BCG-GFP) and Mycobacterium tuberculosis H37Rv were cultured in Middlebrook 7H9 broth (Becton Dickinson) supplemented with 10% albumin dextrose catalase (ADC). Five-day differentiated cells were stimulated with lipopolysaccharide (LPS, 100 ng/mL, Sigma), M. tuberculosis (MOI 5, 10) for 48 hours, BCG-GFP (MOI 10) for different time periods, or fluorescent red latex beads (2-µm diameter, Sigma-Aldrich) for 1.5 hours. After stimulation, cells were collected and fixed with 1% PFA overnight at +4°C. Phagocytosis ratios (BCG-GFP+ or latex bead-PE+ cells) were determined using an Accuri C6 (Becton Dickinson) cytometer.Tumor necrosis factor α (TNF-α), interleukin 10 (IL-10), and CCL2 levels in supernatants were detected with enzyme-linked immunosorbent assay (ELISA) kits (eBiosciences).
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