The protein binding to immune cells was analyzed by immunofluorescence microscopy, as previously reported [31 (link)]. Briefly, PBLs were plated in a cell culture dish (NEST, USA) for 1.5 h, followed by incubation with 5% skim milk for 1 h. After extensive washing with PBS, 50 μg/mL HMGB1, HMG20A, or Trx was added to the PBLs, and the cells were incubated at 22 °C for 2 h. The proteins that bound to PBLs were recognized by a mouse anti-His tag antibody and an Alexa Fluor Plus 488 labeled goat anti-mouse IgG antibody. The cells were fixed with 4% paraformaldehyde and incubated with 4, 6-diamino-2-phenylindole (DAPI) for 10 min. The images were taken with a confocal microscope (Carl Zeiss, Oberkochen, Germany).
Cell culture dish
Manufactured by NEST Biotechnology
Sourced in United States, China
A cell culture dish is a flat, circular container made of transparent material, typically plastic, used for the in vitro cultivation of cells. It provides a controlled environment for the growth and maintenance of cell cultures.
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Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Beyotime (2 mentions)
Confocal microscope, by Zeiss (1 mentions)
Carl lsm710, by Zeiss (1 mentions)
Alexa fluor 488 conjugated goat anti mouse secondary antibody, by Abcam (1 mentions)
Alexa fluor 488 conjugated goat anti mouse igg antibody, by Abcam (1 mentions)
Lsm 710 confocal microscope, by Zeiss (1 mentions)
Paraformaldehyde (pfa), by Biosharp (1 mentions)
Lsm 980, by Zeiss (1 mentions)
Confocal imaging system, by Zeiss (1 mentions)
Confocal laser scanning microscopy, by Zeiss (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Digital eclipse a1 plus microscope, by Nikon (1 mentions)
Dapi staining, by Merck Group (1 mentions)
Ix73 microscope imaging system, by Olympus (1 mentions)
9 protocols using cell culture dish
1
Immunofluorescence Analysis of Protein Binding
2
Immunocytochemical Analysis of Hemocyte Binding
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The immunocytochemical assay was conducted as previously described [28 (link)] with some modification. Briefly, hemocytes collected from healthy shrimp were suspended in the 5% BSA containing resuspending solution [29 (link)], seeded in a cell culture dish (NEST, Wuxi, China) for 3 h, and incubated with rMBP-HSSP-His (50 μg/mL) at room temperature. The same concentration of rMBP-His was used as the negative control. The hemocytes were fixed on dishes with 4% paraformaldehyde prepared in PBS (pH 7.4) for 15 min at room temperature, and washed three times in TBS (50 mM, Tris–HCl, 150 mM NaCl, pH 7.4). After blocking in 5% BSA in TBS at 4 °C overnight, the hemocytes were incubated with mouse anti-MBP antibody (ABclonal, Wuhan, China) in a dilution of 1:500 at room temperature for 1 h, and then incubated with Alexa Fluor 488-conjugated goat anti-mouse secondary antibody (Abcam, Shanghai, China) at a dilution of 1:1000 at room temperature for 1 h. Hemocytes were dyed with Di1 (Beyotime, Shanghai, China) for 20 min and DAPI (Beyotime, Shanghai, China) for 5 min before the observation under a laser confocal scanning microscopy (Carl Zeiss LSM 710, Carl Zeiss, Oberkochen, Germany).
3
Interaction of CsCKM with Peripheral Blood Leukocytes
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The interaction between rCsCKM-1/2 and PBLs were performed as reported previously (35 (link)). Briefly, 1 ×106 cells in 100 μl PBS were settled on the cell culture dish (NEST Biotechnology, Wuxi, China). The dish was blocked with 5% BSA for 1 h and washed with PBS for three times. Then cells were incubated with rCsCKM-1/2 (42.5 μg/ml) or rTrx (42.5 μg/ml) for 1 h and washed as above. Mouse anti-His tag antibody (1:1000 dilution) was added to the dish, and the dish was incubated for 1 h and washed. Goat anti-mouse IgG Alexa Flour 488 (1:2000 dilution) was added to the dish, and the dish was incubated and washed as above. The cells were stained with DAPI and observed with a confocal microscope (Carl Zeiss, Oberkochen, Germany).
4
Visualization of Recombinant Protein Binding
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Binding of recombinant proteins to PBLs was detected by immunofluorescence microscopy as reported previously (44 (link)) with slight adjustments. Briefly, Japanese flounder PBLs were resuspended in L-15 medium to 1×107 cells/ml, and the cells were added to cell culture dish (NEST, USA) for 4 h to allow the cells to settle. The cells were then blocked with 5% skim milk and incubated at 22°C for 1 h. After washing with PBS, 1 μM rPoCXCL10, 1 μM rPoCXCL10M, or PBS were added to the dish and incubated at 22°C for 2 h, followed by washing with PBS for three times. The cells were treated with mouse anti-Flag antibody (ABclonal, Wuhan, China) at 1/1,000 dilution for 1h and then treated with Alexa Fluor® 488-conjugated goat anti-mouse IgG antibody (Abcam, UK) at 1/2,000 dilution for 1 h, followed by washing with PBS for three times. The cells were fixed with 4% paraformaldehyde (Biosharp, Shanghai, China) for 30 min and washed as above, then the cells were stained with 1, 1′-dioctadecyl-3, 3, 3, 3′-tetramethylindocarbocyanine perchlorate (DiI) (Invitrogen, Carlsbad, CA, USA) and 4’, 6-diamidino-2-phenylindole (DAPI) (Beyotime, Shanghai, China). The cells were then observed with a Zeiss LSM 710 confocal microscope (Carl Zeiss, Oberkochen, Germany).
5
Evaluating Mitochondrial Membrane Potential in EBL Cells
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To detect the mitochondrial membrane potential of EBL cells, the mitochondrial probe JC-1 was used according to the manufacturer's instructions (Beyotime, C2006). EBL cells were seeded into the glass bottom CellCulture Dish (Nest, China) and then incubated with 0, 0.25, 0.50 or 1.00 μg/ml GcvH protein. The mitochondrial electron transport chain inhibitor CCCP (50 μM) provided in the kit was used as a positive control and blank cells were used as a negative control. After discarding the cell culture medium, EBL cells were fixed with 4% PFA for 30 min at RT and washed 3 thrice with PBS. 0.5 ml DMEM complete medium and 0.5 ml JC-1 working solution were added and incubated at 37˚C for 20 min. The cells were washed thrice with 1 ml JC-1 staining buffer (1×) and resuspended in 1 ml of DMEM complete medium to observe the amount of red and green fluorescence by the laser confocal microscopy (Zeiss, LSM980).
6
Wnt1 and SRA Immunofluorescence Assay in THP-1 Cells
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THP-1 cells were cultured in the cell culture dish (NEST, USA). After different treatments, the cells were fixed and permeabilized then incubated with anti-wnt1 or anti-SRA antibody (1:100) at 37 °C for 1 h, after washed with PBS and stained with goat-anti-rabbit rhodamine IgG or goat-anti-mouse FITC IgG (Kangwei, China) (1:100) at 37 °C for 1 h, followed by DAPI staining (guge, CHINA). The cells were examined using a Zeiss Confocal Imaging System (Carl Zeiss, Germany).
7
Plasmid Transfection and Confocal Imaging
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After seeded on cell culture dish (NEST, China) for 8 h, HepG2 cells were transfected with GFP-LC3 expression vector (kindly provided by MengYu Liu, Department of Occupational Health, Third Military Medical University, Chongqing 400,038, China) at the final concentration of 1 μg/μL for 4 h using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Samples were then analyzed using laser confocal scanning microscopy (Zeiss, Germany).
8
Cellular Uptake of CDDP-loaded Nanoparticles
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3 × 104 HSC3 cells were added in the cell culture dish (35mm, NEST Biotechnology, Jiangsu, China). After 24 hrs, the cells were incubated with CECMa (equiv. [CDDP] = 5 μM) in the dark for 0,1, 2, 4, 6, 8, and 12 hrs. After that, the cells were washed with PBS 3 times and fixed in 4% paraformaldehyde for 20 min. Pictures of cells were taken by the Nikon Digital Eclipse A1 Plus microscope (Tokyo, Japan) after stained with DAPI. To detect the differential uptake and efficacy with or without LDLR targeting, cells incubation with CECM or CECMa for 2 hrs under normoxia and hypoxia were conduction. ImageJ software was utilized to analyze the fluorescence intensity.
9
Immunocytochemistry of MYC and FLAG
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Macrophages were collected from mice as indicated and seeded in a cell culture dish (NEST, USA). The cells were xed and permeabilized at 4°C for 30 min. After incubation with an anti-MYC antibody (1:2000, CST, #2276, USA) and anti-FLAG antibody (1:2000, CST, #14793, USA) at 4°C overnight, the cells were washed with PBS twice and stained with goat-anti-rabbit FITC-labelled IgG or goat-anti-mouse rhodaminelabelled IgG (1:200, Proteintech, China) at 4°C for 2 h, followed by DAPI staining (Sigma-Aldrich, USA).
The cells were viewed using an Olympus IX73 Microscope Imaging System (Olympus, Japan).
The cells were viewed using an Olympus IX73 Microscope Imaging System (Olympus, Japan).
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