PE-Cy™7 Mouse Anti-Human CD28

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	100 μL of EDTA‐anticoagulated pleural fluid samples were immunophenotyped using six combinations of monoclonal antibodies and a direct immunofluorescence stain	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
100 μL of EDTA‐anticoagulated pleural fluid samples were immunophenotyped using six combinations of monoclonal antibodies and a direct immunofluorescence stain
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.

Publication protocol

A six‐color flow cytometric immunophenotyping study of the pleural fluid was carry out. Briefly, approximately 100 μL of EDTA‐anticoagulated pleural fluid samples were immunophenotyped using six combinations of monoclonal antibodies and a direct immunofluorescence stain‐and‐then‐lyse technique—(Fluorescein isothiocyanate (FITC)/phycoerythrin (PE)/peridin chlorophyll protein cyanin 5.5 (PerCP‐Cy5.5)/PE‐cyanin 7 (PE‐Cy7)/allocycocyanin (APC)/APC‐cyanin 7(APC‐Cy7): 1CD7/CD3/CD5/CD8/CD4/CD45; 2 Kappa/Lambda/CD19/‐/CD45/CD20; 3 CD38/CD138/CD19/CD56/CD45/CD20; 4 CD38/CD30/HLA‐DR/CD10/CD45/CD20; 5 CD38/cyCD79a/CD19/CD10/CD45/CD20; 6 CD38/CD138/CD117/CD28/CD45/CD81; and 7 cyKappa/cyLambda/CD19/CD56/CD45/CD38. (Monoclonal antibodies clones and companies are described in Table 1). 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.

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