Journal: Frontiers in Cell and Developmental Biology

Article Title: Molecular and epigenetic alterations in normal and malignant myelopoiesis in human leukemia 60 (HL60) promyelocytic cell line model

doi: 10.3389/fcell.2023.1060537

Figure Lengend Snippet: Differentiation of HL60 model system using two different culturing conditions. (A) Schema of HL60 myeloid differentiation model into monocytes and granulocytes using vitamin D3 (Vit D3) and all-trans retinoic acid (ATRA) respectively. (B) Bar plot shows mean fluorescence intensity (MFI) of CD14-APC-H7 and CD11b-FITC at 6, 12, 24, 48 and 72 h of induction time points with Vit D3 and ATRA (** = 0.01, *** = 0.001, **** = 0.0001). An unpaired t -test was performed taking a 6 h data point as a reference using GraphPad Prism version 8.2.1. (C) Contour plots showing gating of stained cells with IgG-APC-H7, followed by staining uninduced cells, and Vit D3 induced HL60 cells (72 h induction) with CD14-APC H7 (from left to right contour plots). The X -axis and Y -axis of the contour plots show CD14-APC H7 and forward scatter (A = area) respectively. (D) Bar plots representing MFI of the CD14-APC H7 intensity at 72 h post-Vit D3 induction. The MFI of CD14-APC H7 positive cells were compared with unstained cells (UNS), IgG, and uninduced cells (UI) (* = ≤0.05, ** = 0.01). An unpaired t -test was performed using GraphPad Prism version 8.2.1. (E) Similar to panel C, HL60 cells were induced with ATRA for 72 h and IgG-FITC and CD11b-FITC were used in this experiment. The X -axis and Y -axis of the contour plots show CD11b-FITC and forward scatter (A = area) respectively. (F) Bar plots representing MFI of the CD11b-FITC intensity at 72 h post-ATRA induction. The MFI of CD11b-FITC positive cells were compared with unstained cells (UNS), IgG, and uninduced cells (UI) (* = ≤0.05, ** = 0.01). A statistical test was performed as mentioned in panel D (G) Bar graphs represent the percentage of CD14 positive cells and MFI of CD14-APC H7 positive HL60 cells at 0 and 72 h in RPMI with 10% FBS and IMDM with 20% FBS. The induced cells were compared with their respective uninduced cells in both the culturing conditions in the HL60 differentiation in the IMDM and RPMI media conditions (** = 0.01, *** = 0.001). A statistical test was performed as mentioned in panel (D) (H) Similar to panel G but induction was carried out with ATRA for 72 h. (I) May Grünwald Giemsa staining showing nuclear morphology changes upon HL60 differentiation into monocytes and granulocytes in IMDM (upper panel) and RPMI (lower panel) culturing and induction conditions. The image is taken in Olympus IX83 inverted microscope using CellSens imaging software and 40× magnification.

Article Snippet: The American Type Culture Collection (ATCC) recommends the use of IMDM supplemented with 20% FBS for HL60 cell culture ( https://www.atcc.org/products/ccl-240 ), while the European Collection of Authenticated Cell Cultures (ECACC) recommends RPMI 1640 + 2 mM glutamine supplemented with 10% FBS.

Techniques: Fluorescence, Staining, Inverted Microscopy, Imaging, Software