Flow cytometry Anti-bodies Mouse - CD68

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

CD68 antibody | FA-11

Bio-Rad Laboratories

Upstream tips	Protocol tips	Downstream tips
	Single-cell suspensions were incubated with a Fc receptor block (1 μg/1 × 106 cells; BD Bioscience, San Diego, CA) to reduce nonspecific anti-body binding. Dead cells were excluded using a Live/Dead Fixable Blue Dead Cell Stain Kit (Invitrogen) or 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) (Sigma-Aldrich, St. Louis, MO).	Flow cytometry was performed using BD LSR II and BD FACS Aria III flow cytometers (BD Bioscience), and data were analyzed with FlowJo software (TreeStar, Ashland, OR).

Protocol tips
Single-cell suspensions were incubated with a Fc receptor block (1 μg/1 × 106 cells; BD Bioscience, San Diego, CA) to reduce nonspecific anti-body binding. Dead cells were excluded using a Live/Dead Fixable Blue Dead Cell Stain Kit (Invitrogen) or 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) (Sigma-Aldrich, St. Louis, MO).
Downstream tips
Flow cytometry was performed using BD LSR II and BD FACS Aria III flow cytometers (BD Bioscience), and data were analyzed with FlowJo software (TreeStar, Ashland, OR).

Manufacturer protocol Publication protocol 1 Relevant paper

APC anti-mouse CD68 Antibody

BioLegend

Upstream tips	Protocol tips	Downstream tips
	Mouse ears were subjected to enzymatic digestion by dispase (1 mg/ml; STEMCELL Technologies), collagenase II (2 mg/ml; PAA), and DNase I (0.8 mg/ml, Roche) in PBS for 2 h at 1400 rpm shaking and 37°C. After digestion, the samples were filtered with a 40-μm cell strainer (BD), washed with PBS, and stained with the indicated Abs.	Cell samples were analyzed with a 10-laser flow cytometer (Gallios; Beckman Coulter), and data were analyzed with the Kaluza software (version 1.2; Beckman Coulter).

Protocol tips
Mouse ears were subjected to enzymatic digestion by dispase (1 mg/ml; STEMCELL Technologies), collagenase II (2 mg/ml; PAA), and DNase I (0.8 mg/ml, Roche) in PBS for 2 h at 1400 rpm shaking and 37°C. After digestion, the samples were filtered with a 40-μm cell strainer (BD), washed with PBS, and stained with the indicated Abs.
Downstream tips
Cell samples were analyzed with a 10-laser flow cytometer (Gallios; Beckman Coulter), and data were analyzed with the Kaluza software (version 1.2; Beckman Coulter).

Manufacturer protocol Publication protocol 1 Relevant paper

Purified anti-mouse CD68 Antibody

BioLegend

Upstream tips	Protocol tips	Downstream tips
	Marrow was flushed from the femur of mice immediately after sacrifice in FACS buffer (PBS with 2%FBS, 0.5 mM EDTA). Each stain included 106 cells with anti-mouse F4/80 (Biolegend, or Abcam, A3-1) or anti-mouse CD68 (Biolegend, FA-11). Isotype controls were used to confirm specificity. Flow cytometry was performed using a FACS AriaIII (BD Biosciences).

Protocol tips
Marrow was flushed from the femur of mice immediately after sacrifice in FACS buffer (PBS with 2%FBS, 0.5 mM EDTA). Each stain included 106 cells with anti-mouse F4/80 (Biolegend, or Abcam, A3-1) or anti-mouse CD68 (Biolegend, FA-11). Isotype controls were used to confirm specificity. Flow cytometry was performed using a FACS AriaIII (BD Biosciences).

Manufacturer protocol Publication protocol 1 Relevant paper

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