Comprehensive Bone Marrow Cell Immunophenotyping

The detailed protocol for whole BM staining has been previously reported (Basso-Ricci et al., 2017 (link)). In brief, Precision Count beads (BioLegend) were added to 100 μl of P1 BM sample to allow absolute quantification of hematopoietic cell subsets, and red blood cell lysis was performed. The lysed sample was labeled with fluorescent antibodies for CD3-BV605, CD56-PE-Cy5, CD14-BV510, CD61/41-PE-Cy7, CD135-PE, CD34-BV421, CD45RA-APC-Cy7 (BioLegend), CD33-BB515, CD66b-BB515, CD38-BUV737, CD45-BUV395, CD90-APC, CD10-BV786, CD11c-BV650, CD19-APCR700, CD7-PE-Cy5.5, and CD71-BV711 (BD Biosciences). Titration assays were performed to assess the best antibody concentration. After surface marking, the cells were incubated with PI (BioLegend) to stain dead cells. All samples were acquired using a BD LSR Fortessa (BD Bioscience) flow cytometer after calibration with SPHERO rainbow calibration particles (Spherotech), and raw data were collected using BD FACSDIVA software. The data were subsequently analyzed with FlowJo software, and the graphical output was automatically generated using GraphPad Prism.

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Lam M.T., Coppola S., Krumbach O.H., Prencipe G., Insalaco A., Cifaldi C., Brigida I., Zara E., Scala S., Di Cesare S., Martinelli S., Di Rocco M., Pascarella A., Niceta M., Pantaleoni F., Ciolfi A., Netter P., Carisey A.F., Diehl M., Akbarzadeh M., Conti F., Merli P., Pastore A., Levi Mortera S., Camerini S., Farina L., Buchholzer M., Pannone L., Cao T.N., Coban-Akdemir Z.H., Jhangiani S.N., Muzny D.M., Gibbs R.A., Basso-Ricci L., Chiriaco M., Dvorsky R., Putignani L., Carsetti R., Janning P., Stray-Pedersen A., Erichsen H.C., Horne A., Bryceson Y.T., Torralba-Raga L., Ramme K., Rosti V., Bracaglia C., Messia V., Palma P., Finocchi A., Locatelli F., Chinn I.K., Lupski J.R., Mace E.M., Cancrini C., Aiuti A., Ahmadian M.R., Orange J.S., De Benedetti F, & Tartaglia M. (2019). A novel disorder involving dyshematopoiesis, inflammation, and HLH due to aberrant CDC42 function. The Journal of Experimental Medicine, 216(12), 2778-2799.

Publication 2019

Precision Count beads CD3-BV605 CD56-PE-Cy5 CD14-BV510 CD135-PE CD34-BV421 CD45RA-APC-Cy7 CD38-BUV737 CD45-BUV395 CD90-APC CD10-BV786 CD11c-BV650 CD19-APCR700 BD LSR Fortessa SPHERO rainbow calibration particles

Antibody Assays Cd66b Cd71 Cd90 Cells Cy5 5 Fluorescent antibodies Hematopoietic Prism Red blood cell Stain Titration

Corresponding Organization : Bambino Gesù Children's Hospital

Other organizations : Istituto Superiore di Sanità, University of Rome Tor Vergata, The San Raffaele Telethon Institute for Gene Therapy, Sapienza University of Rome, Rice University, National Institute of Health, Max Planck Institute of Molecular Physiology, Oslo University Hospital, University of Oslo, Karolinska University Hospital, Karolinska Institutet, University of Bergen, Vita-Salute San Raffaele University, San Raffaele University of Rome

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Variable analysis

independent variables

Precision Count beads (BioLegend) added to 100 μl of P1 BM sample
Fluorescent antibodies used for labeling: CD3-BV605, CD56-PE-Cy5, CD14-BV510, CD61/41-PE-Cy7, CD135-PE, CD34-BV421, CD45RA-APC-Cy7, CD33-BB515, CD66b-BB515, CD38-BUV737, CD45-BUV395, CD90-APC, CD10-BV786, CD11c-BV650, CD19-APCR700, CD7-PE-Cy5.5, and CD71-BV711

dependent variables

Absolute quantification of hematopoietic cell subsets

control variables

Red blood cell lysis performed
Titration assays performed to assess the best antibody concentration
Samples acquired using a BD LSR Fortessa (BD Bioscience) flow cytometer after calibration with SPHERO rainbow calibration particles (Spherotech)
Data analyzed with FlowJo software
Graphical output automatically generated using GraphPad Prism

controls

Positive control: SPHERO rainbow calibration particles (Spherotech)
Negative control: Not explicitly mentioned

Annotations

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