Briefly, after the medium was discarded, cells were washed twice with PBS and digested with trypsin/0.25% EDTA (Gibco; Thermo Fisher Scientific, Inc.). Fresh growth medium was added to terminate the digestion and cells were collected by centrifugation at 1,000 × g for 5 min at room temperature. The cells were resuspended and washed twice in 1 ml PBS. The cell pellets were resuspended in 100 µl of HEPES containing 0.5% BSA and 2.5 µg/ml biotinylated MALII (Vector Laboratories, Ltd.), and incubated at room temperature for 2 h in the dark. The cells were washed twice with PBS and incubated with 1 µg/ml streptavidin-phycoerythrin (Sigma-Aldrich; Merck KGaA) for 1 h at room temperature in the dark. After washing the stained cells twice with PBS, cell surface MAL II was quantified using a FACSCanto II flow cytometer (BD Biosciences), and analyzed by BD CellQuest Software version 3.3 (BD Biosciences) according to the manufacturer's instructions.
Streptavidin phycoerythrin
Manufactured by Merck Group
Sourced in Germany
Streptavidin-phycoerythrin is a fluorescent label that consists of the protein streptavidin conjugated to the fluorescent dye phycoerythrin. It is commonly used in flow cytometry and other bioanalytical techniques to detect and quantify target molecules that have been labeled with biotin.
Automatically generated - may contain errors
Lab products found in correlation
Genechip rat gene 2.0 st array, by Thermo Fisher Scientific (2 mentions)
Superscript system, by Thermo Fisher Scientific (2 mentions)
Genechip sample cleanup module, by Qiagen (2 mentions)
Genechip scanner 3000, by Thermo Fisher Scientific (2 mentions)
Nanodrop nd 1000, by Thermo Fisher Scientific (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Rna 6000 nano labchip kit, by Agilent Technologies (2 mentions)
Biotinylated malii, by Vector Laboratories (1 mentions)
Cellquest software version 3, by BD (1 mentions)
Facscanto 2 flow cytometer, by BD (1 mentions)
Propidium iodide, by Thermo Fisher Scientific (1 mentions)
Guava expresspro software, by Merck Group (1 mentions)
Guava easycyte flow cytometer, by Merck Group (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Fluidics station 400, by Thermo Fisher Scientific (1 mentions)
Agilent 2100 bioanalyser, by Agilent Technologies (1 mentions)
4 protocols using streptavidin phycoerythrin
1
Quantification of Cell Surface MAL II Expression
2
Aptamer Binding Assay with Flow Cytometry
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Aptamers were synthesized with 3’-end biotin modification and PAGE-grade purity at IBA GmbH or Biospring GmbH. 100 pmol of biotinylated aptamer was added to 1 × 105 cells in 100 μl PBS / BSA 1% (w/v) and incubated for 30 min at 4°C. The cells were washed with binding buffer and incubated with 100 μl PBS / BSA 1% (w/v) including streptavidin phycoerythrin (Sigma-Aldrich) (1:10) for 30 min at 4°C. The cells were washed twice and resuspended in 200 μl PBS / BSA 1% (w/v) containing propidium iodide (Invitrogen) (1:200) for exclusion of dead cells for analysis. For data acquisition and analysis the Guava easyCyte flow cytometer and the Guava Express Pro software (Millipore) were used.
3
Transcriptome Analysis of Rat Gene Expression
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The quality of RNA was determined by using RNA 6000 Nano LabChip Kit and Agilent Bioanalyzer 2100 (Agilent, Palo Alto, CA, USA). Preparation of cRNA was performed according to the protocol provided by Affymetrix (Santa Clara, CA). The total RNA from individual animal was further purified by using RNeasy Mini Kit (Qiagen Inc., Valencia, CA, USA). Total RNA (3 μg) was converted to double-stranded cDNA using SuperScript System (Invitrogen, Carlsbad, CA) and an oligo (dT24) primer containing a T7 RNA polymerase promoter site. Biotin-labeled cRNA was synthesized from cDNA using a labeling kit and purified by using a GeneChip Cleanup Sample Module (Qiagen Inc., Valencia, CA, USA). The yield of the in vitro transcription reaction was determined by product absorbance at 260 nm measured by NanoDrop ND-1000 (NanoDrop Technologies, Inc., Montchanin, DE), and a size of cRNA probes was evaluated by using RNA 6000 Nano LabChip Kit (Agilent, Palo Alto, CA, USA). Fragmented cRNA was used for hybridization to GeneChip® Rat Gene 2.0 ST arrays (Affymetrix). Arrays were washed and stained with streptavidin-phycoerythrin (Merck, Darmstadt, Germany) in Fluidic Station 400 (Affymetrix), according to the standard protocol of the manufacturer. The arrays were scanned by using GeneChip Scanner 3000 (Affymetrix).
4
Agilent Bioanalyzer RNA Profiling
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The quality of RNA was determined by using an RNA 6000 Nano LabChip Kit and Agilent Bioanalyser 2100 (Agilent, Palo Alto, CA, USA). Preparation of cRNA was performed according to the protocol provided by Affymetrix (Santa Clara, CA). The same amounts of total RNA from two animals were pooled and further purified by using an RNeasy Mini Kit (Qiagen Inc. Total RNA (3 μg) derived from each pool (n = 4) was converted to double-stranded cDNA using a SuperScript System (Invitrogen, Carlsbad, CA) and an oligo(dT)24 primer containing a T7 RNA polymerase promoter site. Biotin-labelled cRNA was synthesised from cDNA using a labelling Kit and purified by using a GeneChip Cleanup Sample Module (Qiagen Inc., Valencia, CA, USA). The yield of the in vitro transcription reaction was determined by the product absorbance at 260 nm, as measured by NanoDrop ND-1000 (NanoDrop Technologies, Inc., Montchanin, DE), size of cRNA probes was evaluated by using RNA 6000 Nano LabChip Kit (Agilent, Palo Alto, CA, USA). Fragmented cRNA was used for hybridization to GeneChip® Rat Gene 2.0 ST arrays (Affymetrix). The arrays were washed and stained with streptavidin-phycoerythrin (Merck, Darmstadt, Germany) in Fluidics Station 400 (Affymetrix) according to the standard protocol of the manufacturer. The arrays were scanned by using a GeneChip Scanner 3000 (Affymetrix).
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