Flow cytometry Anti-bodies Mouse - CD200

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

CD200 Monoclonal Antibody (OX90), PE, eBioscience™

eBioscience

Upstream tips	Protocol tips	Downstream tips
For assessment of CD200 and CD200R1, we used anticoagulant in whole blood for a total volume of 200 μl per tube. After lysis by Red Blood Cell Lysis Buffer (Beijing Solarbio Science and Technology Co., Beijing, China), cells were resuspended in PBS.	, tubes were incubated in the dark at room temperature for 30 min. After washing 2 times, fixing, and permeabilization, we added 10 μL CD200-PE (PE anti-mouseCD200, eBioscience, USA) and CD200R1-PE (PE anti-mouseCD200R1, eBioscience, USA), followed by mixing, and incubating for 30 min at room temperature.	After washing, FACS Canto II flow cytometry (BD, USA) was performed.

Upstream tips
For assessment of CD200 and CD200R1, we used anticoagulant in whole blood for a total volume of 200 μl per tube. After lysis by Red Blood Cell Lysis Buffer (Beijing Solarbio Science and Technology Co., Beijing, China), cells were resuspended in PBS.
Protocol tips
, tubes were incubated in the dark at room temperature for 30 min. After washing 2 times, fixing, and permeabilization, we added 10 μL CD200-PE (PE anti-mouseCD200, eBioscience, USA) and CD200R1-PE (PE anti-mouseCD200R1, eBioscience, USA), followed by mixing, and incubating for 30 min at room temperature.
Downstream tips
After washing, FACS Canto II flow cytometry (BD, USA) was performed.

Manufacturer protocol Publication protocol 1 Relevant paper

Mouse CD200 R3 PE-conjugated Antibody

R&D Systems

Upstream tips	Protocol tips	Downstream tips
	Each cell type was single cell suspended and, incubated in FACS buffer containing pre-conjugated antibodies (see supplementary Table 2) for 20 min on ice. Following incubation, cells were washed twice with FACS buffer.	All the flow cytometry performed using a Cell Lab Quanta SCMPL. The data were analyzed using Kaluza®1.2 software (Beckman Coulter, Denmark) and the expression of each CD markers on the cells was calculated based on the percentage and mean fluorescence intensity (MFI).

Protocol tips
Each cell type was single cell suspended and, incubated in FACS buffer containing pre-conjugated antibodies (see supplementary Table 2) for 20 min on ice. Following incubation, cells were washed twice with FACS buffer.
Downstream tips
All the flow cytometry performed using a Cell Lab Quanta SCMPL. The data were analyzed using Kaluza®1.2 software (Beckman Coulter, Denmark) and the expression of each CD markers on the cells was calculated based on the percentage and mean fluorescence intensity (MFI).

Manufacturer protocol Publication protocol 1 Relevant paper

CD200 antibody | OX-90

Bio-Rad Laboratories

Upstream tips	Protocol tips	Downstream tips
	Cells were incubated with antibodies in 0.1 M PBS (pH7.4) supplemented with 1% FCS and 0.1% sodium azide on ice for 30 minutes. Cells were then washed three times and fixed in 1% paraformaldehyde followed by flow cytometry analysis.	Data were analyzed using the flowjo software (Tree Star, Inc., OR).

Protocol tips
Cells were incubated with antibodies in 0.1 M PBS (pH7.4) supplemented with 1% FCS and 0.1% sodium azide on ice for 30 minutes. Cells were then washed three times and fixed in 1% paraformaldehyde followed by flow cytometry analysis.
Downstream tips
Data were analyzed using the flowjo software (Tree Star, Inc., OR).

Manufacturer protocol Publication protocol 1 Relevant paper

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