Flow cytometry Anti-bodies Mouse - IFN-γ

Flow cytometry is an immunophenotyping technique whereby sing cell suspensions are stained for either cell surface markers or intracellular proteins by fluorescently-labelled antibodies and analyzed with a flow cytometer, where fluorescently-labelled molecules are excited by the laser to emit light at varying wavelengths, which is then detected by the instrument. There are several key criteria which are required to be kept in mind while designing a flow experiment- 1. Antibody titration (optimal dilution of antibodies should be calculated in order to avoid over- or under- saturated signals for proper detection of surface and intracellular markers), 2. Precision (3 or more replicates of the sample should be used per experiment), 3. Specificity (proper isotype controls should be included in the experiment), 4. Day-to-day variability (experiments should be repeated 3 or more times to ensure consistency and avoid variability due to flow cytometer settings), 5. Antibody interaction (Fluorescence minus one or FMO should be used, which is the comparison of signals from panel minus one antibody vs. the full panel), and 6. Antibody stability (fluorescently-labelled antibodies should be stored at 4C).

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Found 3 matching solutions for this experiment

APC Rat Anti-Mouse IFN-γ

BD Biosciences

Upstream tips	Protocol tips	Downstream tips
Spleens and tumors were excised from the 4T1 tumor-bearing mice. Single cell suspensions were produced by homogenizing the spleen samples, and the tumors were minced and subsequently digested with 500 U/mL collagenase type IV (Sigma) for 1 h at 37 °C with agitation. The resulting single cell suspensions were suspended in RPMI 1640 with 10% fetal calf serum (FCS, Gibco, England).	All staining reactions were performed in a final volume of 100 μl at 4 °C. Data were acquired using a FACS Calibur flow cytometer (BD Biosciences, San Diego, CA, USA) and analyzed using FlowJo v7.6.2 software (Tree Star Inc., Ashland, OR, USA).

Upstream tips
Spleens and tumors were excised from the 4T1 tumor-bearing mice. Single cell suspensions were produced by homogenizing the spleen samples, and the tumors were minced and subsequently digested with 500 U/mL collagenase type IV (Sigma) for 1 h at 37 °C with agitation. The resulting single cell suspensions were suspended in RPMI 1640 with 10% fetal calf serum (FCS, Gibco, England).
Protocol tips
All staining reactions were performed in a final volume of 100 μl at 4 °C. Data were acquired using a FACS Calibur flow cytometer (BD Biosciences, San Diego, CA, USA) and analyzed using FlowJo v7.6.2 software (Tree Star Inc., Ashland, OR, USA).

Manufacturer protocol Publication protocol 1 Relevant paper

Brilliant Violet 650™ anti-mouse IFN-γ Antibody

BioLegend

Upstream tips	Protocol tips	Downstream tips
Isolation of splenocytes was done by mashing the spleen through a 70-μm cell strainer. The cells were washed with phosphate-buffered saline and after spinning at 300g for 6 minutes, the cell pellet was resuspended in 1 mL lysis buffer for 3 minutes. Afterward, the cells for cytokine staining were washed with RPMI-1640 +10% fetal calf serum; 1 × 106 cells/well were plated in a 24-well plate and activated with stimulation cocktail for 4 hours.	After fixation and permeabilization cells were stained for the transcription factors (FixPerm True-Nuclear Transcription Factor Buffer Set, 424401; BioLegend).

Upstream tips
Isolation of splenocytes was done by mashing the spleen through a 70-μm cell strainer. The cells were washed with phosphate-buffered saline and after spinning at 300g for 6 minutes, the cell pellet was resuspended in 1 mL lysis buffer for 3 minutes. Afterward, the cells for cytokine staining were washed with RPMI-1640 +10% fetal calf serum; 1 × 106 cells/well were plated in a 24-well plate and activated with stimulation cocktail for 4 hours.
Protocol tips
After fixation and permeabilization cells were stained for the transcription factors (FixPerm True-Nuclear Transcription Factor Buffer Set, 424401; BioLegend).

Manufacturer protocol Publication protocol 1 Relevant paper

Purified Rat Anti-Mouse IFN-γ

BD Biosciences

Upstream tips	Protocol tips	Downstream tips
	Cells were stained with fixable viability dye eF780 or eF506 (eBioscience). ). All cells were fixed and permeabilized by the FOXP3 Fixation/Permeabilization Concentrate and Diluent kit (eBioscience).

Protocol tips
Cells were stained with fixable viability dye eF780 or eF506 (eBioscience). ). All cells were fixed and permeabilized by the FOXP3 Fixation/Permeabilization Concentrate and Diluent kit (eBioscience).

Manufacturer protocol Publication protocol 1 Relevant paper

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