Flow cytometry Anti-bodies Human - CD138

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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APC Mouse Anti-Human CD138

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. CD30‐PE was purchased from Beckman Coulter and all other antibodies were purchased from BD Biosciences.

Protocol 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. CD30‐PE was purchased from Beckman Coulter and all other antibodies were purchased from BD Biosciences.

Protocol 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. CD30‐PE was purchased from Beckman Coulter and all other antibodies were purchased from BD Biosciences.

Manufacturer protocol Publication protocol 1 Relevant paper

PE Mouse Anti-Human CD138

BD Biosciences

Upstream tips	Protocol tips	Downstream tips
	, approximately 100 μL of EDTA‐anticoagulated pleural fluid samples were immunophenotyped using six combinations of monoclonal antibodies and a direct immunofluorescence.	The cells were acquired in a FACSCanto flow cytometer (Becton Dickinson, San Jose, CA) using the FACSDiva software (Becton Dickinson). A minimum of 100,000 events were acquired. For data analysis the Infinicyt software (Cytognos SL, Salamanca, Spain) was used.

Protocol tips
, approximately 100 μL of EDTA‐anticoagulated pleural fluid samples were immunophenotyped using six combinations of monoclonal antibodies and a direct immunofluorescence.
Downstream tips
The cells were acquired in a FACSCanto flow cytometer (Becton Dickinson, San Jose, CA) using the FACSDiva software (Becton Dickinson). A minimum of 100,000 events were acquired. For data analysis the Infinicyt software (Cytognos SL, Salamanca, Spain) was used.

Manufacturer protocol Publication protocol 1 Relevant paper

CD138 Antibody, anti-human, APC

Miltenyibiotec

Upstream tips	Protocol tips	Downstream tips
	To analyse the cell survival of isolated BMMCs, cells were transferred, centrifugation at 1500 g for 10 min and incubated with antibodies for 20 min at 37 °C. Annexin V was then added and stained for 15 min at room temperature. After incubation, BMMCs were washed, centrifuged, measured by a FACSCanto II (BD Biosciences) and resulting data were analysed using FlowJo software.

Protocol tips
To analyse the cell survival of isolated BMMCs, cells were transferred, centrifugation at 1500 g for 10 min and incubated with antibodies for 20 min at 37 °C. Annexin V was then added and stained for 15 min at room temperature. After incubation, BMMCs were washed, centrifuged, measured by a FACSCanto II (BD Biosciences) and resulting data were analysed using FlowJo software.

Manufacturer protocol Publication protocol 1 Relevant paper

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