About 1000 DB, Daudi, or a 1:1 mixture of cells were diluted into 1 mL of artificial cerebrospinal perfusion fluid (aCSF; Harvard Apparatus) which closely matched the fluidic properties (viscosity, salt concentrations) of CSF. Samples were introduced to the device at the flow rate of 2 mL/hr. Following the cell capture, fix/perm buffer was perfused over the cells for 10 min, followed by permSB for 5 min, and PBS containing 2% FBS and 1% BSA for 5 min, all at a flow rate of 1 mL/hr. A cocktail of antibodies containing 1 µL of anti-Ki-67, anti-CD19, and anti-CD20, and 2 µL of anti-κ light chain and anti-λ light chain was perfused over the cells at 1 mL/hr for 5 min. Lastly, to reduce background signal from antibodies binding to the channel surface, washing buffer (PBS with 2% FBS and 1% BSA) was perfused at 1 mL/hr for 5 min. Alternatively, cells were exposed to Ibrutinib-BFL, followed by staining with Hoechst 33342 and anti-CD20-APC (clone 2H7; BioLegend). Images were captured on a Nikon Eclipse TE2000S inverted microscope (Nikon) equipped with four filter sets (#31000v2, #41001, #41002b, #41024; Chroma Technology).
Anti cd20 apc clone 2h7
Manufactured by BioLegend
Anti-CD20-APC (clone 2H7) is a fluorescently labeled antibody that binds to the CD20 antigen expressed on the surface of B lymphocytes. The APC (Allophycocyanin) fluorescent dye is conjugated to the antibody.
Automatically generated - may contain errors
Lab products found in correlation
Eclipse te2000 s inverted microscope, by Nikon (1 mentions)
Hoechst 33342, by BioLegend (1 mentions)
Facsjazz cell sorter, by BD (1 mentions)
Lightcycler 96 instrument, by Roche (1 mentions)
Rhamp snp genotyping system, by Integrated DNA Technologies (1 mentions)
Annexin 5 fitc, by Thermo Fisher Scientific (1 mentions)
Streptavidin apc, by Thermo Fisher Scientific (1 mentions)
Zombie nir viability dye, by BioLegend (1 mentions)
Anti cd45 clone 30 f11, by BioLegend (1 mentions)
Anti f4 80 clone bm8, by BioLegend (1 mentions)
Anti cd47 clone b6h12, by BioXCell (1 mentions)
Quanteon flow cytometer, by FlowJo (1 mentions)
Anti cd11b clone m1 70, by BioLegend (1 mentions)
3 protocols using anti cd20 apc clone 2h7
1
Immunophenotyping of Leukemia Cells
2
Tracking Donor Chimerism Using SNPs
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We identified SNPs that distinguished the donor and recipient MCMs using a panel of 12 SNPs (see Additional file 3:Table S2 ) and the rhAMP SNP Genotyping System (Integrated DNA Technologies) as described previously38 (link),75 (link), giving preference to homozygous/homozygous mismatches. We performed the rhAMP assays in triplicate using a LightCycler 96 Instrument (Roche Molecular Systems) in the endpoint analysis mode.
We quantified donor chimerism in whole PBMCs or immune subsets by sequencing the diagnostic SNPs with an Illumina MiSeq using previously described primer sets, PCR conditions, and analysis pipeline (see also Additional file 4:TableS3 and Additional file 5:Table S4 )38 (link). To isolate specific immune subsets, we stained PBMCs with anti-CD3 FITC (clone SP34; BD Pharmingen), anti-CD14 PerCP-Cy5.5 (clone M5E2; BioLegend), anti-CD20 APC (clone 2H7; BioLegend), anti-CD45 PE (clone D058-1283; BD Pharmingen), and Live/Dead Fixable Near-IR vital dye (ThermoFisher Scientific) and sorted into monocyte (singlets, live, lymphocytes, CD45mid, CD14+ cells), T lymphocyte (singlets, live, lymphocytes, CD14−, CD45hi, CD3+), or B lymphocyte (singlets, live, lymphocytes, CD14−, CD45hi, CD20+) populations using a BD FACSJazz cell sorter. We used between 179 and 61,251 cells for gDNA extraction and sequencing for each sorted population.
We quantified donor chimerism in whole PBMCs or immune subsets by sequencing the diagnostic SNPs with an Illumina MiSeq using previously described primer sets, PCR conditions, and analysis pipeline (see also Additional file 4:Table
3
Flow Cytometric Profiling of Ramos Cells
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