Orai1 surface expression was analyzed by flow cytometry. To this end, the cells were detached, washed three times with phosphate-buffered saline (PBS) and fixed with 4 % paraformaldehyde for 15 min on ice without (for surface protein) or with (for total protein) permeabilization with 0.1 % Triton X-100 for 5 min. Then the cells were incubated for 60 min (37 °C) with anti-Orai1 primary antibody (1:200, Abcam), washed once in PBS, and stained in 1:250 diluted CF™ 488A-labeled anti–rabbit secondary antibody (Sigma, USA) for 30 min (37 °C). Samples were immediately analyzed on a FACS Calibur flow cytometer (BD Biosciences).
Cf 488a labeled anti rabbit secondary antibody
Manufactured by Merck Group
Sourced in United States
The CF™ 488A-labeled anti-rabbit secondary antibody is a laboratory reagent designed for use in various immunoassay techniques. It is a goat-derived antibody that has been conjugated with the fluorescent dye CF™ 488A, which has excitation and emission spectra suitable for detection with common fluorescence instrumentation.
Automatically generated - may contain errors
Lab products found in correlation
Facscalibur flow cytometer, by BD (4 mentions)
Anti orai1 primary antibody, by Abcam (3 mentions)
Facscalibur, by BD (1 mentions)
Glass chamber slides, by Sarstedt (1 mentions)
Lsm 5 exciter confocal laser scanning microscope, by Zeiss (1 mentions)
Plan neofluar 40 1.3 na dic, by Zeiss (1 mentions)
Draq 5 dye, by Biostatus (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Orai1 rabbit anti mouse antibody, by Abcam (1 mentions)
6 protocols using cf 488a labeled anti rabbit secondary antibody
1
Quantifying Orai1 Surface Expression
2
Orai1 Expression Analysis by Flow Cytometry
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Orai1 expression was analyzed by flow cytometry. Cultured cells were detached, washed three times with phosphate-buffered saline (PBS), and fixed with 4% paraformaldehyde for 15 min on ice. The cells were then incubated for 60 min (37 °C) with anti-Orai1 monoclonal primary antibody (1:200, Abcam) washed once in PBS, and stained in 1:250 diluted CF™ 488A-labeled anti-rabbit secondary antibody (Sigma) for 30 min (37 °C). Samples were immediately analyzed on a FACSCalibur flow cytometer (BD Biosciences, Heidelberg, Germany). Data were analyzed using the FlowJo software (FlowJo LLC, Ashland, Oregon, USA).
3
COX-2 Expression Analysis by Flow Cytometry
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Cyclooxygenase-2 (COX-2) expression was analyzed by flow cytometry. Cultured cells were detached, washed three times with phosphate-buffered saline (PBS) and fixed with 4 % paraformaldehyde for 15 min on ice. Then the cells were incubated for 60 min (37 °C) with anti-COX-2 primary antibody (1:200, #ab23672, Abcam), washed once in PBS, and stained in 1:250 diluted CF™ 488A-labeled anti-rabbit secondary antibody (Sigma) for 30 min (37 °C). Samples were immediately analyzed on a FACS Calibur flow cytometer (BD Biosciences, Heidelberg, Germany). In parallel cultures, the supernatants were also collected and soluble 6-keto-PGF1a levels (# ab141709; Abcam) were measured using an Enzyme-linked immunosorbent assay (ELISA). Soluble levels in the cell culture media were determined according to the manufacturer's protocol.
4
Orai1 Localization in Chorein-Silenced Cells
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For confocal laser scanning microscopy negative and chorein silenced ZF cells were seeded on glass chamber slides (Sarstedt, Germany). After washing twice with PBS, cells were fixed with 4% PFA for 15 min and blocked with 3% BSA in PBS for 1 hour at room temperature. Then, the cells were exposed to anti-Orai1 primary antibody (1:200, Abcam) at 4 °C overnight. After three washing steps with PBS the cells were incubated with CF™ 488A-labeled anti-rabbit secondary antibody (1:250, Sigma, USA) and with DRAQ-5 dye (1:3000, Biostatus, Leicestershire, UK) for 1 h at room temperature. Following three washes with PBS all slides were mounted with ProLong Gold antifade reagent (Life Technologies, USA). Images were subsequently taken on a Zeiss LSM 5 EXCITER confocal laser scanning microscope (Carl Zeiss, Germany) with a water immersion Plan-Neofluar 40/1.3 NA DIC [14, 34] .
5
Quantifying Orai1 Surface Expression
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To determine Orai1 surface abundance, negative and chorein silenced ZF cells were detached, washed two times with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde for 20 min at room temperature. Then the cells were blocked for 30 min with 3% BSA in PBS and incubated for 60 minutes (37°C) with anti-Orai1 primary antibody (1:100, Abcam). After two washes with PBS the cells were stained with a CF™ 488A-labeled anti-rabbit secondary antibody (1:200, Sigma, USA) for 30 minutes (37°C). After two washing steps samples were analyzed on a FACS Calibur flow cytometer (BD Biosciences).
6
Orai1 Surface Expression in Platelets
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Orai1 surface expression was analyzed in platelets by flow cytometry. Washed platelets were incubated with 2 µg/ml CRP without BSA, and subsequently fixed with 1% paraformaldehyde for 30 minutes on ice. After rinsing three times, platelets were incubated for 90 minutes with Orai1 rabbit anti-mouse antibody (Abcam), washed once in Tyrode buffer, and stained in 1:250 diluted CF™ 488A-labeled anti-rabbit secondary antibody (Sigma, USA) for 60 minutes. Samples were immediately analyzed on a FACS Calibur flow cytometer (BD Biosciences).
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