HEK293 cells were purchased from American Type Culture Collection (Manassas, VA). These cells were cultured in high glucose Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% foetal bovine serum, 2 mM L-glutamine, 100 IU/ml penicillin and 100 µg/ml streptomycin. Serum-free media HCELL-100 was purchased from WISENT (St-Bruno, Canada). Poly-L-lysine coated coverslips were purchased from Corning (Bedford, MA). Anti-V5, Anti-V5 HRP and Anti-V5 FITC-linked monoclonal antibodies were purchased from Invitrogen (Waltham, MA). HRP-linked Anti-GAPDH rabbit monoclonal antibody was purchased from Cell Signaling Technology (Danvers, MA). Goat polyclonal anti-Hemojuvelin antibody and t-butoxycarbonyl-Gln-Ala-Arg-7-amino-4-methylcoumarin (Boc-QAR-AMC) were purchased from R&D Systems (Minneapolis, MN). Lipofectamine 3000 was purchased from Invitrogen (Carlsbad, CA). Centrifugal filters were purchased from Merck Millipore (Cork, Ireland). Lysis buffer (1% Triton, 50 mM Tris, 150 mM NaCl, 5 mM EDTA) was supplemented with protease inhibitor from Roche (Mannheim, Germany). Protein A/G PLUS-agarose beads were purchased from Santa-Cruz Biotechnology (Dallas, TX).
Poly l lysine coated coverslips
Manufactured by Corning
Sourced in United States
Poly-L-lysine coated coverslips are a type of lab equipment used for cell culture applications. The coverslips are coated with the positively charged amino acid polymer poly-L-lysine, which enhances cell adhesion and attachment to the surface.
Automatically generated - may contain errors
Lab products found in correlation
Duolink in situ detection reagents orange, by Merck Group (2 mentions)
A1r confocal microscope, by Nikon (2 mentions)
Prolong gold antifade reagent with dapi, by Cell Signaling Technology (2 mentions)
Aurora b, by Abcam (2 mentions)
Alexa fluor 647 phalloidin, by Thermo Fisher Scientific (2 mentions)
Lamin b, by Proteintech (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Centrifugal filter, by Merck Group (1 mentions)
Hek293 cell, by American Type Culture Collection (1 mentions)
Protein a g plus agarose bead, by Santa Cruz Biotechnology (1 mentions)
Hcell 100, by Wisent (1 mentions)
Hrp linked anti gapdh rabbit monoclonal antibody, by Cell Signaling Technology (1 mentions)
T butoxycarbonyl gln ala arg 7 amino 4 methylcoumarin boc qar amc, by R&D Systems (1 mentions)
Cos 7 cells, by American Type Culture Collection (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Transit lt transfection reagent, by Mirus Bio (1 mentions)
Goat anti rabbit igg af 647, by Thermo Fisher Scientific (1 mentions)
Prolong gold with dapi, by Thermo Fisher Scientific (1 mentions)
Rat anti mouse mhc 2 clone 2g9, by BD (1 mentions)
12 protocols using poly l lysine coated coverslips
1
Culturing and Analyzing HEK293 Cells
2
Overexpression of HOMER2 Variants in Cells
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HEK293T cells and COS7 cells (ATCC, Manassas, VA, USA) were grown in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% FBS (Life Technologies, Carlsbad, CA, USA). Cells were incubated in a 5% CO2-humidified incubator at 37°C. Cells were grown on Poly-L-Lysine Coated coverslips (Corning, Tewksbury, MA, USA). Clonal cells were obtained by transfection with Transit-LT Transfection Reagent (Mirus Bio, Madison, WI USA) using cMYC-tagged HOMER2WT and FLAG-tagged HOMER2p.Arg185Pro plasmid constructs according to the manufacturer’s instructions.
3
Immunofluorescent Staining of Mouse MHC-II
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Skin cell suspensions were obtained by Liberase digestion as mentioned above. Cell suspensions were diluted in RPMI and deposited in microplates containing poly-L-lysine-coated coverslips (Corning). Cells were incubated overnight at 4°C and fixed with 4% paraformaldehyde. Cells were then incubated with rat anti-mouse MHC-II (clone 2G9, BD Bioscience) and rabbit anti-clathrin heavy chain (Abcam), followed by AF647 goat anti-rabbit IgG (Invitrogen) and AF-555 goat anti-rat IgG (Invitrogen). Finally, coverslips were mounted on microscope slides using ProLong Gold with DAPI (Life Technologies). Cells were visualized with a LMS 700 confocal microscope (Zeiss) and pictures were edited using Zen software (Zeiss).
4
Immunofluorescence Staining of Dendritic Cells
5
Protein-Protein Interaction Visualization
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Cells were cultured for 24 h on poly-L-lysine coated coverslips (Corning) in 24-well plates. Cells with or without cellular stress induction were fixed and permeabilized with ice-cold 100% methanol. PLA reaction was carried out according to the manufacturer’s instructions using Duolink in situ Detection Reagents (Millipore Sigma). Briefly, coverslips were blocked using Duolink Blocking Solution (Millipore Sigma) for 1 h at 37 °C, followed by incubation with mouse anti-FLAG and goat anti-HSP27 antibodies overnight at 4 °C. Coverslips were washed by 1 × Wash Buffer (Millipore Sigma) three times the next day, and were subsequently incubated with the PLA probes (PLUS-probe-coupled anti-mouse and MINUS-probe-coupled anti-goat antibodies) (Millipore Sigma). Duolink in situ Detection Reagents Orange (Millipore Sigma) were used for ligation and signal amplification. ProLong Gold Antifade Reagent with DAPI (Cell Signaling Technology) was used to stain nuclei and to prevent the fading of fluorescence. In this procedure, bright fluorescent dots were observed when two probes are in close proximity. Fluorescence signals were visualized with a Nikon A1R+ confocal microscope (Nikon, Melville, NY, USA).
6
Visualizing Daam1-Mediated Spirochete Engulfment
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Cell infection assays and immunofluorescent stainings were performed in duplicate for each cell line, timepoint, and treatment as described above with the following modifications. Cells were seeded in 6 well cell culture plates containing poly-L-lysine coated coverslips (Corning) for 24 hr. Cell culture medium was replaced with fresh media containing 10 μg rabbit polyclonal anti-Daam1 antibody (Proteintech) with incubation for 10 min followed by addition of GFP-expressing WT B. burgdorferi to the cells at a MOI of 100, and incubated at 37°C with 5% CO2. Cells were fixed with methanol at -20°C for 20 min after 1 and 2 hr post-infection. After fixation, coverslips were blocked (Pierce Protein Free T20 Blocking Buffer TBS) for 30 mins at room temperature. Cells were stained for Daam1, and mounted onto glass slides using ProLong Diamond Antifade with DAPI (Molecular Probes by Life Technologies). Slides were viewed using a Zeiss LSM 800 confocal laser scanning microscope to count the number of coiling events detected (Daam-1 pseudopods enwrapping a spirochete) in 1000 total neuroglial cells per coverslip.
7
Quantifying Invadopodia-Mediated Extracellular Matrix Degradation
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The procedure was performed as described elsewhere8 (link). The purpose of the experiment was to assess the number of active invadopodia digesting fluorescently-labeled gelatin in cells. Poly-L-Lysine coated coverslips (Corning) placed in a 24 well plate and rinsed with 1 × PBS were fixed by incubation with 0.5% glutaraldehyde for 15 min at RT, followed by another washing with PBS. The coverslips were immediately placed on a drop of gelatin-fluorescein (Invitrogen) and incubated in the dark for 10 min at RT. To quench residual glutaraldehyde, coverslips were incubated in 5 mg/mL sodium borohydride solution at RT, followed by washing with PBS. Before seeding the cells, the coverslips were rinsed with a cold cell culture medium. Thirty thousand cells were seeded per well, onto the side of coverslip coated with gelatin-fluorescein in a 24 well plate in a full cell culture medium and kept at 37 °C, 5% CO2 for 12 h before fixing with 4% FA. The cells were stained using phalloidin-Alexa Fluor 568 and Hoechst 33342 (Invitrogen) to visualize F-actin and cell nuclei, respectively. The images were taken on a confocal microscope. The number of invadopodia and the area of gelatin digestion was estimated by using the ImageJ software. The total number of cells per clone, which were analyzed, was 10.
8
Proximity Ligation Assay for Protein Interactions
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Cells were cultured for 24 h on poly-L-lysine coated coverslips (Corning) in 24-well plates. Cells with or without cellular stress induction were fixed and permeabilized with ice-cold 100% methanol. PLA reaction was carried out according to the manufacturer’s instructions using Duolink In Situ Detection Reagents Orange (MilliporeSigma). ProLong Gold Antifade Reagent with DAPI (Cell Signaling Technology) was used to stain nuclei and to prevent fading of fluorescence. Fluorescence signals were visualized with a Nikon A1R+ confocal microscope (Nikon).
9
Quantifying Neutrophil Extracellular Trap Formation
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To further assess NETs formation in vitro, neutrophils were seeded on poly-L-lysine-coated coverslips (#354085, Corning, Inc.) and stimulated with PMA for 3 h and then fixed with 4% paraformaldehyde (PFA, #BL539A, Biosharp) for 20 min at room temperature, rinsed twice in PBS, incubated in 50 mM of NH4Cl for 10 min at room temperature and permeabilized with 0.5% Triton X-100 (#BB151-500, Thermo Fisher Scientific, Inc.) for 1 min at room temperature. The cells were then blocked in PBS containing 1% bovine serum albumin (BSA, #A3294, Sigma-Aldrich; Merck KGaA) for 30 min at 37°C and incubated with neutrophil elastase (NE) antibody (#sc-55549, Santa Cruz Biotechnology, Inc.) at 1:400 dilution in blocking buffer for 1 h at room temperature. Following washes 3 times in PBS, the cells were incubated in the presence of fluorochrome-conjugated secondary antibodies (#A11001, Alexa Fluor 488, Invitrogen; Thermo Fisher Scientific, Inc.) at 1:200 dilution at room temperature for 1 h, rinsed twice in PBS, stained with Hoechst 33342 (#C1022, Beyotime Institute of Biotechnology) at 1:5 dilution at room temperature for 5 min, rinsed in PBS, and the coverslips were mounted onto glass slides using a fluorescence microscope (DM400B, Leica Microsystems GmbH). The percentage of NETs was evaluated by counting the number of NET-releasing neutrophils out of the total number of neutrophils.
10
Fluorescent Gelatin Degradation Assay
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Fluorescent-gelatin degradation assays were performed according to the protocol described by Mazurkiewicz et al. [29 (link)]. Poly-L-lysine-coated coverslips (Corning) were washed with PBS and fixed with 0.5% glutaraldehyde for 15 min. Further, the coverslips were placed on a 30 μl drop of fluorescein (FITC)-labeled gelatin, incubated for 10 min, and rinsed with PBS. Then, the residual reactive groups were quenched with sodium borohydride (5 mg/ml). Cells were trypsinized, seeded in 24-well plates on glass coverslips, and incubated at 37 °C for 16 h. Next, cells were fixed with 4% formaldehyde, permeabilized with 0.1% Triton X-100, and stained with Alexa Fluor 568 phalloidin to visualize filamentous actin. Leica SP8 confocal microscope with LAS X software (ver. 3.3.0, Leica) was used for image capture. Places of degraded gelatin were visible as dark holes (the absence of fluorescence) in the green, fluorescent gelatin matrix.
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