Chronic lymphocytic leukemia B-cell-derived TNFα impairs bone marrow myelopoiesis

Abstract

CLL patient BM HSPCs exhibit aberrant molecular and functional characteristics CLL B-cell-derived TNFa upregulates PU.1 and GATA-2 in BM HSPCs The effects of CLL B-cellderived TNFa are reversible upon TNFa neutralization Chronic TNFa exposure in vitro recapitulates ex vivo HSPC functional deficiencies

Standardized flow-cytometry-based protocol to simultaneously measure transcription factor levels

“…However, following testing and determination of the staining protocol to use, no deviations from that protocol are allowed to maintain the integrity of the standardization. The protocol presented below reflects that which was used in ( Manso et al., 2021 ) and ( Manso et al., 2019 ) for freshly isolated, primary human bone marrow cells. Note: Keep all reagents and cells on ice (4 ° C) unless otherwise indicated.…”

“… This includes both the full stain panel and the paired isotype control panel. The author’s utilized 5x10 6 cells per individual stain (10x10 6 total for stain and isotype control panels) as they were targeting rare cell populations (see ( Manso et al., 2021 )). Note that this number can be adjusted to the user’s requirements, but should remain constant throughout the entire use of this protocol to maintain standardization.…”

“… Dilute stock FVD to the manufacturer’s recommended dilution (refer to the manufacturer’s technical data sheet). Example: the FVD used in ( Manso et al., 2021 ) and ( Manso et al., 2019 ) required a 1:1000 dilution, adding 1 μL stock FVD for every 1 mL 1 × PBS. Note: Fixable viability dyes work by staining free amine groups found in proteins.…”

“…The protocol below describes the specific steps for using freshly isolated or cultured cells to determine cellular levels of transcription factors. Here, we describe the universal procedure the authors utilized to determine transcription factor levels in hematopoietic stem and progenitor cells (HSPCs) from human bone marrow as found in ( Manso et al., 2021 ) and ( Manso et al., 2019 ). However, this protocol can be adapted to any cell type and experimental model species (such as mouse) assuming the specific reagents required are available for that cell type and/or species.…”

“…This protocol uses bone marrow cells but can be adapted for other cell types. For complete details on the use and execution of this protocol, please refer to Manso et al. (2021) and Manso et al.…”