Flow cytometry Anti-bodies Mouse - MHCII

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

MHC Class II (I-A/I-E) Monoclonal Antibody (M5/114.15.2), FITC, eBioscience™

eBioscience

Upstream tips	Protocol tips	Downstream tips
For all experiments, cells were incubated in 0.5 μg Fc Block (BD Biosciences) for 10 minutes at RT. Surface staining was performed in the dark for 30 minutes at 4 °C in staining buffer. Cells were then washed twice with staining buffer followed by fixation in 1% paraformaldehyde (VWR, West Chester, PA, USA)		For flow cytometry immunophenotyping experiments, cells were acquired on an LSR II cytometer (BD Immunocytometry Systems, San Jose, CA, USA) equipped with 405 nm, 488 nm, 561 nm, and 640 nm excitation lasers. The spleen FACS experiments were performed using a FACSAria II instrument (BD Immunocytometry Systems) equipped with 405 nm, 488 nm, or 633 nm lasers located at the University of Chicago Flow Cytometry Core Facility, Chicago, IL, USA. All data collection and sorting were performed using BD FACS Diva software (BD Biosciences) and data analyses were performed using FlowJo software (Tree Star, Ashland, OR, USA).

Upstream tips
For all experiments, cells were incubated in 0.5 μg Fc Block (BD Biosciences) for 10 minutes at RT. Surface staining was performed in the dark for 30 minutes at 4 °C in staining buffer. Cells were then washed twice with staining buffer followed by fixation in 1% paraformaldehyde (VWR, West Chester, PA, USA)
Downstream tips
For flow cytometry immunophenotyping experiments, cells were acquired on an LSR II cytometer (BD Immunocytometry Systems, San Jose, CA, USA) equipped with 405 nm, 488 nm, 561 nm, and 640 nm excitation lasers. The spleen FACS experiments were performed using a FACSAria II instrument (BD Immunocytometry Systems) equipped with 405 nm, 488 nm, or 633 nm lasers located at the University of Chicago Flow Cytometry Core Facility, Chicago, IL, USA. All data collection and sorting were performed using BD FACS Diva software (BD Biosciences) and data analyses were performed using FlowJo software (Tree Star, Ashland, OR, USA).

Manufacturer protocol Publication protocol 1 Relevant paper

MHC Class II (I-A/I-E) Monoclonal Antibody (M5/114.15.2), eFluor 450, eBioscience™

eBioscience

Upstream tips	Protocol tips	Downstream tips
	For cell-surface antigen staining, the samples were incubated with antibodies for 20 min at 4 °C, washed and resuspended in PBS containing 3% FBS. To exclude dead cells, 7-amino actinomycin D (7-AAD, BD Biosciences) was added to the samples before sorting. We sorted live, single cells using a FACSAria III Cell Sorter with FACSDiva ver. 8.0.1 (BD Biosciences).

Protocol tips
For cell-surface antigen staining, the samples were incubated with antibodies for 20 min at 4 °C, washed and resuspended in PBS containing 3% FBS. To exclude dead cells, 7-amino actinomycin D (7-AAD, BD Biosciences) was added to the samples before sorting. We sorted live, single cells using a FACSAria III Cell Sorter with FACSDiva ver. 8.0.1 (BD Biosciences).

Manufacturer protocol Publication protocol 1 Relevant paper

APC/Cyanine7 anti-mouse I-A/I-E Antibody

BioLegend

Upstream tips	Protocol tips	Downstream tips
	Single cell suspensions were stained with fluorescently conjugated antibodies in a 1:100 dilution unless otherwise noted. Cells were stained with LIVE/DEAD™ Aqua or Blue (Invitrogen), blocked with 4μg/ml anti-CD16/32 (2.4G2; Bioxcell)	Samples were analyzed on an LSRII and sorted on an Aria II cell sorter (BD Biosciences).

Protocol tips
Single cell suspensions were stained with fluorescently conjugated antibodies in a 1:100 dilution unless otherwise noted. Cells were stained with LIVE/DEAD™ Aqua or Blue (Invitrogen), blocked with 4μg/ml anti-CD16/32 (2.4G2; Bioxcell)
Downstream tips
Samples were analyzed on an LSRII and sorted on an Aria II cell sorter (BD Biosciences).

Manufacturer protocol Publication protocol 1 Relevant paper

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