293FT/ACE2/TMPRSS2/DSP1-7 cells in 24-well plates were treated with the indicated concentrations of the compounds for 2 days. Cells were harvested using Cellstripper (Corning) and fixed with 4% formaldehyde before incubation with either anti-ACE2 antibody (Proteintech) or mouse IgG1 isotype control (BioLegend) antibody at 4°C. Cells were stained with PE-conjugated secondary antibody (Thermo Fisher Scientific) and analyzed using the FACSCanto II instrument (BD Biosciences, San Jose, CA, USA). Data were analyzed using the FlowJo software (Tree Star Inc., San Carlos, CA, USA).
Pe conjugated secondary antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
PE-conjugated secondary antibody is a laboratory reagent used in immunoassays to detect and quantify target proteins or other biomolecules. It consists of a secondary antibody conjugated to the fluorescent dye phycoerythrin (PE), which can be excited by a light source and emit a detectable signal. This product provides a standardized and reproducible tool for researchers to amplify and visualize specific molecular interactions in their experiments.
Automatically generated - may contain errors
Lab products found in correlation
Trypsin edta, by Thermo Fisher Scientific (3 mentions)
Mouse igg1 isotype control, by BioLegend (2 mentions)
Anti ace2 antibody, by Proteintech (2 mentions)
Facstar plus flow cytometer, by BD (2 mentions)
Facscanto 2, by BD (1 mentions)
Cellstripper, by Corning (1 mentions)
Prolong gold dapi antifade reagent, by Thermo Fisher Scientific (1 mentions)
Anti cd3ε clone 2c11, by Thermo Fisher Scientific (1 mentions)
Soluble anti cd28 clone 37, by Thermo Fisher Scientific (1 mentions)
Rtk4530, by BioLegend (1 mentions)
Recombinant il 12, by Thermo Fisher Scientific (1 mentions)
Fv3000 confocal laser scanning microscope, by Olympus (1 mentions)
Lsrfortessa, by BD (1 mentions)
Facs canto 2 instrument, by BD (1 mentions)
Cd11b, by Thermo Fisher Scientific (1 mentions)
Foxp3 staining buffer set, by Thermo Fisher Scientific (1 mentions)
Dulbecco s phosphate buffered saline, by Thermo Fisher Scientific (1 mentions)
Fluorescein isothiocyanate (fitc), by BD (1 mentions)
Accutase, by STEMCELL (1 mentions)
Ab8295, by Abcam (1 mentions)
10 protocols using pe conjugated secondary antibody
1
ACE2 and TMPRSS2 Expression in 293FT Cells
2
Cytokine Profiling of Activated CD4+ T Cells
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Colonic-LP CD45+CD3+CD4+CD44low cells were sorted and seeded into 96-well plates for stimulation with plate-bound anti-CD3ε (clone 2C11, 5 µg/ml, eBioscience) and soluble anti-CD28 (clone 37.51, 2 µg/ml, eBioscience) antibodies alone or in combination with recombinant IL-12 (100 ng/ml, Peprotech) in complete RPMI medium and were incubated for 8 h at 37°C. After incubation, supernatants were collected for cytokine ELISA detection.
For STAT4 staining, CD4+ T cells were incubated at 37°C for 5 h in complete RPMI medium alone or with recombinant IL-12 (100 ng/ml, Peprotech) and then fixed (4% PFA) for 30 min at room temperature. The cells were then rinsed three times in PBS for 5 min and incubated in a blocking buffer (0.1% Saponin in 1% BSA–PBS) for 30 min at room temperature. The cells were then incubated for 2 h at room temperature with purified anti-STAT4 (15A1B41, Biolegend) and IgG2b isotype (RTK4530, Biolegend) antibodies diluted in a blocking buffer, followed by two washes in a blocking buffer for 5 min and incubated overnight at 4°C. The following day, the cells were incubated for 1 h with a PE-conjugated secondary antibody (Thermo Scientific), then washed and mounted with Prolong gold DAPI antifade reagent (Molecular Probes).
For STAT4 staining, CD4+ T cells were incubated at 37°C for 5 h in complete RPMI medium alone or with recombinant IL-12 (100 ng/ml, Peprotech) and then fixed (4% PFA) for 30 min at room temperature. The cells were then rinsed three times in PBS for 5 min and incubated in a blocking buffer (0.1% Saponin in 1% BSA–PBS) for 30 min at room temperature. The cells were then incubated for 2 h at room temperature with purified anti-STAT4 (15A1B41, Biolegend) and IgG2b isotype (RTK4530, Biolegend) antibodies diluted in a blocking buffer, followed by two washes in a blocking buffer for 5 min and incubated overnight at 4°C. The following day, the cells were incubated for 1 h with a PE-conjugated secondary antibody (Thermo Scientific), then washed and mounted with Prolong gold DAPI antifade reagent (Molecular Probes).
3
Quantification of HIF-1α in Irradiated Mice
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Twenty four hours after irradiation, mice were sacrificed. Tumors were fixed in 4% paraformaldehyde and embedded in paraffin. The blocks were cut into 5-μm-thick sections. Antigen retrieval was accomplished at 37°C with protease K solution. For immunofluorescence staining, deparaffinized sections were blocked with 10% normal horse serum for 1 hour and then incubated with primary antibodies against HIF-1α for overnight at 4°C (Abcam). After washing with PBS, the samples were incubated for 1 hour with a PE conjugated secondary antibody (Thermo Fisher Scientific, Waltham, MA, USA). Reactions showed under the fluorescence microscope, and HIF-1α stained cells were quantified using Image-J software.
4
Chondrocyte Extracellular Matrix Analysis
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Immunofluorescence was performed to explore the changes in the expression of aggrecan, collagen type II, MMP3 and MMP13. Chondrocytes were incubated in blocking solution and stained with primary antibodies against aggrecan, collagen type II, MMP3 and MMP13 overnight at 4°C. Chondrocytes were then stained with the FITC- or phycoerythrin (PE)-conjugated secondary antibody (Thermo Fisher). The nucleus was labelled with Hoechst 33,342. Cells were observed under an Olympus FV3000 confocal laser scanning microscope.
5
CD45 and PD-L1 Expression Analysis
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Ba/F3 murine pro-B cell line and human peripheral blood mononuclear cells (PBMC) were used as positive and negative controls for mouse CD45 expression (VioGreen-CD45 130-110-665, Miltenyi Biotec, Gladbach, Germany), respectively. For PD-L1 staining, cells were incubated with PD-L1 antibody (1:100, 30 min at room temperature) (#PA5-28115, Thermo Fisher Scientific, MA, USA), washed and resuspended in PBS and marked with PE-conjugated secondary antibody (#31864, Thermo Fisher Scientific, MA, USA) before analysis (BD LSR Fortessa, BD Biosciences, San Jose, CA, USA). Data were analyzed using FlowJo version 10.8.0 (BD Biosciences, San Jose, CA, USA). FACS gating strategies are available in Supplementary Fig. 6 .
6
ACE2 Expression Quantification by Flow Cytometry
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The details of this procedure have been described previously. 6) (link) Cells were fixed with 4% formaldehyde before incubation with either anti-ACE2 antibody (ProteinTech) or mouse IgG1 isotype control (BioLegend) antibody at 4 °C. Cells were stained with PE-conjugated secondary antibody (Thermo Fisher Scientific) and analyzed using the FACSCanto II instrument (BD Biosciences). Data were analyzed using the FlowJo software (Tree Star Inc., San Carlos, CA, U.S.A.).
7
Characterization and Viability of rbHP-PCs
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After trypsinizing and harvesting 80-90% cultured rbHP-PCs, cells were washed with flow cytometric (FC) buffer. We used a 100 μl volume suspension of 1×10 6 cells with a fluorochrome-conjugated antibody: CD11b 1:100, CD90 1:100, CD81 1:100 (Thermo Fisher Scientific) in FC buffer and then cultured it at 4ºC for 30 min. A 100 μl volume suspension of a non-conjugated antibody, primary antibody CD34 1:100, CD29 1:100 (Thermo Fisher Scientific), CD44 1:100 (SeroTec, USA), was also made in FC buffer and cultured it at 4°C for 30 min. Then, a 100 μl volume incubation for a phycoerythrin (PE)-conjugated secondary antibody (1:100) (Thermo Fisher Scientific) was done in FC buffer at 4ºC for 30 min. After washing, 500 μl volume of 2% paraformaldehyde was added to fix the cell and a FC analysis was performed.
Cultured rbHP-PCs were harvested and seeded onto a 96-well plate (1x10 3 cells per well). After 24 hours, 10 ul CCK-8 reagents (Dojindo Laboratory, Kumamoto, Japan) were added and incubated for 2 hours. Then, the supernatant was transferred to a new plate, and the optical density was measured at a 450 nm wavelength.
Cultured rbHP-PCs were harvested and seeded onto a 96-well plate (1x10 3 cells per well). After 24 hours, 10 ul CCK-8 reagents (Dojindo Laboratory, Kumamoto, Japan) were added and incubated for 2 hours. Then, the supernatant was transferred to a new plate, and the optical density was measured at a 450 nm wavelength.
8
Flow Cytometry Analysis of Stem Cell Markers
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The cells were handled as described previously [31 (link)]. Briefly, cells were harvested with Accutase (Stem Cell Technologies, Vancouver, Canada). For the detection of the membrane marker SSEA4, cells were fixed in 1% PFA and stained with FITC (fluorescein isothiocyanate)-conjugated SSEA4 antibody (cat no. 560126, BD Biosciences, San Jose, USA, 1:100) and isotype-matched controls. For the detection of the intracellular antigen OCT4, cells were fixed and permeabilized by Foxp3 Staining Buffer Set (Invitrogen, Carlsbad, CA, USA), blocked in Dulbecco’s phosphate-buffered saline (Gibco, Carlsbad, CA, USA) containing 5% FBS, then stained with primary un-conjugated OCT4 (cat no. ab19857, Abcam, 1:200) and followed by PE-conjugated secondary antibody (1:200; eBioscience, San Diego, USA). The cells incubated with secondary antibody only were used as negative controls. Cells were then analyzed and quantified with the flow cytometry (FACStar Plus Flow Cytometer, Becton-Dickinson, San Jose, CA, USA).
9
Collagen Immunostaining and Proteoglycan Analysis
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Samples were prepared with five-micrometer thickness sections and used for immunocytochemistry (ICC) and Alcian blue 8GX (Roth) staining. The immune staining was performed on deparaffinized sections using Col II and X rabbit anti-human antibodies (Abcam, 1:20 and 1:80 dilutions), respectively. Col stands for collagen. The PE-conjugated secondary antibody (eBioscience, 1:80 dilution, goat anti-rabbit) was used for fluorescence visualization. For proteoglycan production assays, sections were stained with Alcian blue 8GX and examined using Genucam digital camera.
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Samples were prepared with five-micrometer thickness sections and used for immunocytochemistry (ICC) and Alcian blue 8GX (Roth) staining. The immune staining was performed on deparaffinized sections using Col II and X rabbit anti-human antibodies (Abcam, 1:20 and 1:80 dilutions), respectively. Col stands for collagen. The PE-conjugated secondary antibody (eBioscience, 1:80 dilution, goat anti-rabbit) was used for fluorescence visualization. For proteoglycan production assays, sections were stained with Alcian blue 8GX and examined using Genucam digital camera.
10
Purification and Characterization of hiPSCs, hCVPCs, and hCMs
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The hiPSCs, hCVPCs and hCMs analyzed by flow cytometry as previously described [32 (link),33 (link),37 (link)] and incubated with antibodies: PE-conjugated SSEA4 (eBioscience, 12-8843-42, 1:100), PE-conjugated SSEA1 (eBioscience, 12-8813-42, 1:50), or un-conjugated cTnT (Abcam, ab8295, 1:200) and ki67 (Abcam, ab16667, 1:200) with PE-conjugated secondary antibody (eBioscience, 12-4010-87, 1:200) or isotype-matched negative controls. Cells were then analyzed by FACS (FACS tar Plus Flow Cytometer, Becton-Dickinson, San Jose, CA, USA).
Cell sorting was performed using a FACS sorter (Beckman Moflo Astrios) based on the SSEA1 detection as we reported previously [37 (link)]. Briefly, The Mix cells were dissociated 0.05% Trypsin-EDTA (Thermo Fisher Scientific) for 5 min at 37 °C and washed once with the wash buffer (1% FBS in Dulbecco's phosphate-buffered saline, DPBS, Invitrogen, 14190144) after collected. The cells were then incubated with the primary antibody (PE-conjugated SSEA1, eBioscience, 12-8813-42, 1:50) for 1 h at 4 °C after blocking. Then the suspensions were filtered with a 40 μm cell strainer and sorted using Beckman Moflo flow cytometer. The SSEA1+ cells (Mix-hCVPCs) and SSEA1- cells (Mix-hCMs) were collected and used for further analysis.
Cell sorting was performed using a FACS sorter (Beckman Moflo Astrios) based on the SSEA1 detection as we reported previously [37 (link)]. Briefly, The Mix cells were dissociated 0.05% Trypsin-EDTA (Thermo Fisher Scientific) for 5 min at 37 °C and washed once with the wash buffer (1% FBS in Dulbecco's phosphate-buffered saline, DPBS, Invitrogen, 14190144) after collected. The cells were then incubated with the primary antibody (PE-conjugated SSEA1, eBioscience, 12-8813-42, 1:50) for 1 h at 4 °C after blocking. Then the suspensions were filtered with a 40 μm cell strainer and sorted using Beckman Moflo flow cytometer. The SSEA1+ cells (Mix-hCVPCs) and SSEA1- cells (Mix-hCMs) were collected and used for further analysis.
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