Flow cytometry Anti-bodies Human - CD105

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

PE Mouse anti-Human CD105

BD Biosciences

Upstream tips	Protocol tips	Downstream tips
After incubation with monoclonal antibodies for 15 min (min) at 4°C, erythrocytes were lysed with ammonium chloride (PharmLyse, BD Biosciences, San Diego, California) at room temperature for 10 min using a standard lyse/wash technique. Samples were acquired on FACSCanto II instruments (BD Biosciences). For the detection of cytoplasmic antigens, cells were fixed and permeabilized using 4% formaldehyde and 0.25% Saponin.		Data were analyzed using FCS Express software (De Novo Software, Los Angeles, California). Non‐viable cells, debris, and aggregates were excluded based on forward scatter‐height/forward scatter‐area (FSC‐H/FSC‐A).

Upstream tips
After incubation with monoclonal antibodies for 15 min (min) at 4°C, erythrocytes were lysed with ammonium chloride (PharmLyse, BD Biosciences, San Diego, California) at room temperature for 10 min using a standard lyse/wash technique. Samples were acquired on FACSCanto II instruments (BD Biosciences). For the detection of cytoplasmic antigens, cells were fixed and permeabilized using 4% formaldehyde and 0.25% Saponin.
Downstream tips
Data were analyzed using FCS Express software (De Novo Software, Los Angeles, California). Non‐viable cells, debris, and aggregates were excluded based on forward scatter‐height/forward scatter‐area (FSC‐H/FSC‐A).

Manufacturer protocol Publication protocol 1 Relevant paper

Human Endoglin/CD105 APC-conjugated Antibody

R&D Systems

Upstream tips	Protocol tips	Downstream tips
Human MSC (5 × 105 cells) were suspended in PBS and incubated with combinations of the monoclonal antibodies described above and also was acquired hMSC unstained used as control.		We used a FACSCanto II flow cytometer (BD Biosciences San Jose, CA) for EV acquisition using the FACSDiva 6.1 software (BD Biosciences). The FACSCanto II flow cytometer is an 8-color instrument with 3 lasers, blue (488-nm, air cooled, 20-mW solid state), red (633 nm, 17-mW HeNe) and violet (405 nm, 30-mW solid state).

Upstream tips
Human MSC (5 × 105 cells) were suspended in PBS and incubated with combinations of the monoclonal antibodies described above and also was acquired hMSC unstained used as control.
Downstream tips
We used a FACSCanto II flow cytometer (BD Biosciences San Jose, CA) for EV acquisition using the FACSDiva 6.1 software (BD Biosciences). The FACSCanto II flow cytometer is an 8-color instrument with 3 lasers, blue (488-nm, air cooled, 20-mW solid state), red (633 nm, 17-mW HeNe) and violet (405 nm, 30-mW solid state).

Manufacturer protocol Publication protocol 1 Relevant paper

Human Endoglin/CD105 PE-conjugated Antibody

R&D Systems

Upstream tips	Protocol tips	Downstream tips
	Heparinized bone marrow (BM) and peripheral blood (PB) cells (106 leukocytes per tube) were incubated with various combinations of mAb (Supplementary Table S3) for 15 minutes. Then, erythrocytes were lysed in FACS-Lysing-Solution (BD Biosciences, San José, CA, USA). Washed cells were acquired on a FACSCalibur (BD Biosciences) and analyzed by FlowJo software (TreeStar, Ashland, OR, USA)

Protocol tips
Heparinized bone marrow (BM) and peripheral blood (PB) cells (106 leukocytes per tube) were incubated with various combinations of mAb (Supplementary Table S3) for 15 minutes. Then, erythrocytes were lysed in FACS-Lysing-Solution (BD Biosciences, San José, CA, USA). Washed cells were acquired on a FACSCalibur (BD Biosciences) and analyzed by FlowJo software (TreeStar, Ashland, OR, USA)

Manufacturer protocol Publication protocol 1 Relevant paper

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