The cells were fixed with 4% paraformaldehyde buffer for 30 min, permeabilized with 0.5% Triton X-100 for 20 min, and blocked with blocking buffer (Beyotime) for 30 min at room temperature. The cells were probed with LC3, IL-1β, and CD63 antibodies (1:1000 dilution) at 4°C overnight and subsequently detected with anti-rabbit IgG Fab2 Alexa Fluor® 488 molecular probes (1:2000 dilution). After staining the cell nuclei with DAPI (0.1 µg/mL stock solution) for at least 3 min, the immunofluorescent images were obtained using the Leica EL6000 Microscope (Leica Microsystems).
El6000 microscope
Manufactured by Leica
Sourced in United States
The EL6000 is a high-performance microscope designed for laboratory use. It features a powerful illumination system and advanced optics to provide clear, detailed images of specimens. The core function of the EL6000 is to enable detailed observation and analysis of samples.
Automatically generated - may contain errors
Lab products found in correlation
L10382, by Thermo Fisher Scientific (2 mentions)
Blocking buffer, by Beyotime (1 mentions)
Ksf medium, by Thermo Fisher Scientific (1 mentions)
Keratinocyte serum free medium, by Thermo Fisher Scientific (1 mentions)
Chick embryo extract, by Thermo Fisher Scientific (1 mentions)
Igf 1, by R&D Systems (1 mentions)
Basic fibroblast growth factor (bfgf), by R&D Systems (1 mentions)
7 protocols using el6000 microscope
1
Immunofluorescent Staining of Cellular Markers
2
Macrophage Recruitment in Viral Infection
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ANA-1 macrophage cells (labeled with GFP lentiviral vectors from Genechem, Shanghai, China) were cultivated in the upper chamber of a 24-well Transwell plate (104 cells/well). ANA-1 or BEAS-2B cells (labeled with mRFP-LC3 lentiviral vectors) or A549 cells were cultivated in the lower chamber (105 cells/mL). The cells in the lower chambers were infected with 10 TCID50 of the H1N1 virus for 2 h and then transferred to a fresh culture medium. Macrophage recruitment was evaluated by tracking the movement of GFP+ ANA-1 cells under the Leica EL6000 Microscope (Leica Microsystems CMS GmbH).
3
Quantifying Autophagy Levels via LC3 Puncta
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Cells were grown on Lab-Tek™ II Chamber Slide™ System slides. After experimentation, cells were fixed using formalin, permeabilized using 0.5% BSA, 0.2% saponin in a 1×PBS solution, and blocked with 0.5% BSA, in a 1×PBS solution. Samples were probed with rabbit anti-LC3B antibody (L10382, Invitrogen) and a fluorescence conjugated secondary anti-rabbit antibody was then used for visualization. DAPI was used for nuclear counterstaining. LC3 puncta in cells were observed using a Leica EL6000 microscope and analyzed using Leica software. A minimum of 100 cells was examined for the presence of LC3 puncta in each condition of the individual experiments.
4
Quantifying Autophagy Levels via LC3 Puncta
5
Investigating PM10 and P. aeruginosa Effects on HCET Cells
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HCET cells (HCE-2 [50.B1], ATCC, Gaithersburg, MA) were cultured in Keratinocyte-serum free (KSF) medium (Gibco, Grand Island, NY) with 5ng/ml human recombinant EGF, 0.05mg/ml bovine pituitary extract, 0.005mg/ml insulin, and 500ng/ml hydrocortisone as previously described (Somayajulu et al., 2023 (link)). Cells were treated with PM10 (0, 25, 50, 100, 200, 500, 800 and 1200µg/ml for 24h for the MTT assay. For all other experiments, cells were incubated with 100μg/ml (per cell viability data from dose curve; 75-80% viability) PM10 at 37°C and 5% CO2 for 24hr. To investigate the combined effects of PM10 on P. aeruginosa infected cells, a subset of cells were challenged with strain 19660 at a multiplicity of infection (MOI) of 20 for 3h. To assess the effects of SKQ1 (BOC Sciences, Shirley, NY, USA), another subset of cells were incubated with 50nM SKQ1, 1h before PM10 exposure (Somayajulu et al., 2023 (link)) and then challenged with strain19660 at similar MOI. Another group of HCET were challenged with strain 19660 at a MOI of 20 for 3h to assess the effects of bacteria alone on these cells. Phase contrast microscopy was used to photograph cell preparations using a Leica EL 6000 microscope (Deerfield, IL, USA). All the images were acquired at the same magnification and processed similarly.
6
Assessing SKQ1 Effects on Corneal Cells
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HCET cells (HCE-2 [50.B1], ATCC, Gaithersburg, MA, USA) were cultured in Keratinocyte-serum free medium (Gibco, Grand Island, NY, USA) with 5 ng/mL human recombinant EGF, 0.05 mg/mL bovine pituitary extract, 0.005 mg/mL insulin, and 500 ng/mL hydrocortisone as reported before [73 (link)]. HCEC (primary corneal epithelial cells, ATCC) were cultured in Keratinocyte-serum free medium (Gibco) with 5 ng/mL human recombinant EGF and 0.05 mg/mL bovine pituitary extract as per the manufacturer’s protocol. To evaluate the effects of SKQ1, a subset of cells was incubated with 50 nM SKQ1 as previously described [59 (link)], 1 h prior to PM10 exposure. Phase contrast microscopy was used to photograph cell preparations using a Leica EL 6000 microscope (Deerfield, IL, USA).
7
Neurosphere Culture and Measurement
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