APC-H7 Mouse Anti-Human CD43

Protocol tips

Upstream tips	Protocol tips	Downstream tips
		Immediately after sample preparation, a minimum of 5 × 104 events/sample aliquot was collected using a FACSCanto II flow cytometer equipped with the FACSDiva™ software program (BD Biosciences, USA). For data analysis, the Infinicyt™ 1.7 software (Cytognos SL, Salamanca, Spain) was employed.

Downstream tips
Immediately after sample preparation, a minimum of 5 × 104 events/sample aliquot was collected using a FACSCanto II flow cytometer equipped with the FACSDiva™ software program (BD Biosciences, USA). For data analysis, the Infinicyt™ 1.7 software (Cytognos SL, Salamanca, Spain) was employed.

Publication protocol

BM samples were collected in EDTA anticoagulant while aspirates and biopsy samples were placed in saline. Staining of surface markers followed by erythrocyte lysis was performed according to well‐established protocols 12. The following panel of eight‐color combinations of monoclonal antibodies was used for the diagnosis of BCL: CD20+CD4/CD45/CD8+Igλ/CD56+Igκ/CD5/CD19/CD3/CD38 (lymphoid screening tube), CD20/CD45/CD23/CD10/CD200/CD19/CD79b/CD43, CD20/CD45/CD31/LAIR‐1/CD11c/CD19/sIgM/CD81,CD20/CD45/CD103/CD95/CD22/CD19/CXCR5/CD49d, and CD20/CD45/CD62L/CD39/X/CD19/CD27/X. The fluorochrome order was: pacific blue (PacB), pacific orange (PacO), fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridinin chlorophyll protein (PerCP‐Cy5.5), phycoerythrin/Cy7 (PE‐Cy7), allophycocyanin (APC), and allophycocyanin/Cy7 (APC‐Cy7) and “X” means no added marker. Details about technical information on CD39, CD43, CD81, and CD95 reagents can be observed in Table 1. Immediately after sample preparation, a minimum of 5 × 104 events/sample aliquot was collected using a FACSCanto II flow cytometer equipped with the FACSDiva™ software program (BD Biosciences, USA). For data analysis, the Infinicyt™ 1.7 software (Cytognos SL, Salamanca, Spain) was employed. Merge and calculation of flow cytometric data corresponding to each individual sample was performed as previously described 13, 14, using the Infinicyt™ 1.7 software program (Cytognos SL, Salamanca, Spain) after gating on CD19+ and CD20+ lymphoma cells. Whenever identified, residual normal B cells were excluded from analysis. Then, the mean fluorescence intensity (MFI) values of each marker for each individual were obtained and the median value of the MFI of groups with the same neoplasm was calculated. In addition, comparison among groups was performed by multivariate analysis, such as the principal component analysis (PCA), using information on the markers from the eight‐color panel, with the exception of immunoglobulin light chain kappa (Igκ) and lambda (Igλ).

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