The expression of EGFR, HER2, and E-cadherin on the surface of the oral epithelial cells was quantified by flow cytometry. Briefly, OKF6/TERT-2 cells in 6-well tissue culture plates were incubated with tissue culture medium with or without IFN-γ for 24 h and then infected with 5 × 105C. albicans cells. After 75 min, the cells were scraped from the wells with a cell scraper, fixed with 3% paraformaldehyde, blocked with 1% goat serum, and then stained with specific antibodies (for EGFR, sc-101, and for HER2, sc-33684, from Santa Cruz Biotechnology; for E-cadherin, ab1416 from Abcam, Inc.), followed by an Alexa 488-conjugated goat or mouse anti-rabbit antibody (Life Technologies, Inc.). Control epithelial cells were incubated in a similar concentration or mouse or rabbit IgG (Abcam, Inc.). The fluorescence of the cells was determined by flow cytometry, analyzing at least 10,000 cells per condition.
Sc 101
Manufactured by Santa Cruz Biotechnology
The Sc-101 is a laboratory instrument designed to perform high-throughput analysis of biological samples. It is capable of rapid and accurate quantification of target molecules or markers within complex matrices. The core function of the Sc-101 is to provide researchers with a reliable and efficient tool for data acquisition and analysis in various life science applications.
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Lab products found in correlation
3 protocols using sc 101
1
Quantification of EGFR, HER2, and E-cadherin Expression on Oral Epithelial Cells
2
Immunofluorescence Imaging of HIF1A, EGFR, and Phospho-EGFR
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For immunofluorescence imaging, cells grown on glass coverslips (or IBIDI µ-dish 35 mm) were fixed in 4% paraformaldehyde, washed three times with PBS, permeabilised with 0.5% Triton X-100 in PBS for 10 min and washed again three times with PBS. Coverslips were then blocked with 3% BSA in PBS for 1 h, incubated with antibodies against HIF1A (BD Biosciences #610958; dilution: 1/100), EGFR (Santa Cruz #sc-101; dilution: 1/100) and Phospho-EGFR (anti-Tyr1173 Santa Cruz #sc-12351, dilution: 1/50), washed, stained with fluorescent secondary antibodies, washed, counterstained with Hoescht and finally mounted with fluorescent mounting medium (Dako) under a glass coverslip. Images were acquired with a Zeiss LSM 780 NLO laser confocal microscope (Zeiss, Germany). For EGFR and Phospho-EGFR imaging, three stacks of 2 µm were acquired and compiled into a single image by projection of the max intensity of each stack.
3
Localization Imaging of EGFR Variants
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For localization imaging the cells were cultivated on round cover glasses in a 12-well plate at 37 °C. After 24 h the cells were washed in 2× PBS and fixed in 4% formaldehyde ( prepared from paraformaldehyde). After blocking in 1× PBS and 3% BSA for 1 h at RT the cells were incubated with the anti EGFRvIII antibody (L8A4, mouse IgG, 1 µg mL -1 in 3% PBS/ BSA) or with the EGFRwt antibody (Santa Cruz #sc-101, mouse IgG) for 1 h. After washing three times with 1× PBS/0.5% Tween20 the specimens were incubated with the ALEXAfluor® 594 labeled secondary antibody (in 3% BSA, ALEXAfluor® 594 anti-mouse antibody 1 : 1000; Molecular Probes or Thermo Fisher Scientific) for 1 h in the dark followed by washing three times in 1× PBS/0.5% Tween20 and counter-stained with DAPI (1 : 1000). The specimens were embedded in ProlongGold antifade medium and sealed with nail polish. Until image acquisition the specimens were stored at 4 °C in the dark.
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