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2 protocols using apc conjugated cd45ro

1

Phenotypic Analysis of Human PBMCs

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Peripheral blood mononuclear cells (PBMCs) were isolated from fresh blood by density gradient centrifugation on Ficoll-Paque PLUS (Cytiva). The isolated PBMCs were then analyzed with an 8-color flow cytometer (FACSCanto™II, BD Biosciences, CA, USA) after labeling with the following anti-human fluorochrome-conjugated antibodies: FITC-conjugated CD38 (clone HIT2); APC or FITC-conjugated PD-1 (clone EH12.2H7); FITC-conjugated CD45RA (clone HI100); PE-conjugated CD24 (clone ML5); PE-conjugated ICOS (clone C398.4A); PE-conjugated CCR6 (clone G034E3); PerCP-conjugated CD3 (clone UCHT1); PE/cyanine7-conjugated CD27 (clone O323); PE/cyanine7-conjugated CD4 (clone OKT4); APC-conjugated CD19 (clone HIB19); APC-conjugated CD45RO (clone UCHL1); APC-conjugated CXCR3 (clone G025H7); and Brilliant Violet 421-conjugated CXCR5 (clone J252D4) (all antibodies were from BioLegend, CA, USA). Cell viability was assessed using a live/dead assay (Fixable Viability Dye eFluor 780; eBioscience). All data were collected and analyzed using FlowJo software. The gating strategies of the isotype-matched IgG control for specific cell populations are shown in the supplementary figure (Figure S1).
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2

Measuring CAR Expression on T Cells

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To measure CAR expression, cells were first suspended in 100 μL of cold buffer (PBS containing 0.5% BSA and 2 mM EDTA) supplemented with 2 μg of goat IgG (Jackson Immunoresearch, West Grove, PA, USA) and incubated on ice. CD19-FLAG CAR-T cells were stained with 2 μL of PE- or Alexa Fluor 488-conjugated anti-FLAG (Biolegend), whereas BCMA CAR-T cells were first stained with 0.4 μg of BCMA-huFc protein (Acro Biosystems, Newark, DE, USA) and then stained with 1 μL of PE- or Alexa Fluor 488-conjugated goat anti-human IgG (Jackson Immunoresearch). Cells were co-stained with either FITC-conjugated anti-CD4 and APC-conjugated anti-CD8; PE-conjugated anti-CD27 and APC-conjugated CD45RO; or APC-conjugated anti-PD-1 (all from Biolegend). Dead cells were identified with 7-aminoactinomycin D (7-AAD, BioLegend). The cells were rinsed with 3 mL of buffer, then suspended in buffer and acquired on a FACSCalibur (BD Biosciences, San Jose, CA, USA). Gating strategies are shown in Supplemental Figure S1.
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