Flow cytometry Anti-bodies Mouse - TNF-α

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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Purified anti-mouse TNF-α Antibody

BioLegend

Upstream tips	Protocol tips	Downstream tips
	BMDMs were differentiated for 24 hours in M0, M1 or M2 conditions and blocked with anti-mouse FcR antibody (CD16/CD32, BD) for 15 min at 4°C in FACS buffer (PBS with 2% FBS and 1 mM EDTA), subsequently cells were stained for 15 min at 4°C with blue-fluorescent reactive dye. Cells were washed three times with FACS buffer, fixed with the eBioscience Fixation/Permeabilization buffer for 40 min at 4°C and washed three times in 1X eBioscience Permeabilization buffer. For intracellular staining, cells were first blocked with anti-mouse FcR antibody (CD16/CD32, BD) in 1X Permeabilization buffer (eBioscience) for 45 min at 4°C prior to staining.

Protocol tips
BMDMs were differentiated for 24 hours in M0, M1 or M2 conditions and blocked with anti-mouse FcR antibody (CD16/CD32, BD) for 15 min at 4°C in FACS buffer (PBS with 2% FBS and 1 mM EDTA), subsequently cells were stained for 15 min at 4°C with blue-fluorescent reactive dye. Cells were washed three times with FACS buffer, fixed with the eBioscience Fixation/Permeabilization buffer for 40 min at 4°C and washed three times in 1X eBioscience Permeabilization buffer. For intracellular staining, cells were first blocked with anti-mouse FcR antibody (CD16/CD32, BD) in 1X Permeabilization buffer (eBioscience) for 45 min at 4°C prior to staining.

Protocol tips

BMDMs were differentiated for 24 hours in M0, M1 or M2 conditions and blocked with anti-mouse FcR antibody (CD16/CD32, BD) for 15 min at 4°C in FACS buffer (PBS with 2% FBS and 1 mM EDTA), subsequently cells were stained for 15 min at 4°C with blue-fluorescent reactive dye. Cells were washed three times with FACS buffer, fixed with the eBioscience Fixation/Permeabilization buffer for 40 min at 4°C and washed three times in 1X eBioscience Permeabilization buffer. For intracellular staining, cells were first blocked with anti-mouse FcR antibody (CD16/CD32, BD) in 1X Permeabilization buffer (eBioscience) for 45 min at 4°C prior to staining.

Manufacturer protocol Publication protocol 1 Relevant paper

PE-Cy™7 Rat Anti-Mouse TNF

BD Biosciences

Upstream tips	Protocol tips	Downstream tips
	Cells were blocked using 0.5 µg anti-CD16/32 antibody for 10 min at 4°C and then stained with anti-TCR γδ mAb for 30 min at 4°C, followed by washing with PBS and fixing in 4% paraformaldehyde. Stained cells were permeabilized using 0.1% saponin (Sigma-Aldrich; Merck KGaA) and incubated with anti-IFN-γ and anti-TNF-α for 30 min at 4°C.	Stained cells were immediately analyzed using the FACSCanto™ II flow cytometer (BD Immunocytometry Systems; BD Biosciences). Data were analyzed using FACSDiva™ 2.0 software (BD Immunocytometry Systems; BD Biosciences).

Protocol tips
Cells were blocked using 0.5 µg anti-CD16/32 antibody for 10 min at 4°C and then stained with anti-TCR γδ mAb for 30 min at 4°C, followed by washing with PBS and fixing in 4% paraformaldehyde. Stained cells were permeabilized using 0.1% saponin (Sigma-Aldrich; Merck KGaA) and incubated with anti-IFN-γ and anti-TNF-α for 30 min at 4°C.
Downstream tips
Stained cells were immediately analyzed using the FACSCanto™ II flow cytometer (BD Immunocytometry Systems; BD Biosciences). Data were analyzed using FACSDiva™ 2.0 software (BD Immunocytometry Systems; BD Biosciences).

Manufacturer protocol Publication protocol 1 Relevant paper

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