Characterization of neutrophils and macrophages from ex vivo-cultured murine bone marrow for morphologic maturation and functional responses by imaging flow cytometry

Abstract

Neutrophils and macrophages differentiate from common myeloid progenitors in the bone marrow, where they undergo nuclear morphologic changes during maturation. During this process, both cell types acquire critical innate immune functions that include phagocytosis of pathogens, and for neutrophils the release of nuclear material called nuclear extracellular traps (NETs). Primary cells used to study these functions are typically purified from mature mouse tissues, but bone marrow-derived ex vivo cultures provide more abundant numbers of progenitors and functionally mature cells. Routine analyses of these cells use conventional microscopy and flow cytometry, which present limitations; microscopy is laborious and subjective, whereas flow cytometry lacks spatial resolution. Here we describe methods to generate enriched populations of neutrophils or macrophages from cryopreserved mouse bone marrow cultured ex vivo, and to use imaging flow cytometry that combines the resolution of microscopy with flow cytometry to analyze cells for morphologic features, phagocytosis, and NETosis.

Keywords: Cell morphology; Fluorescence microscopy; Myeloid; NETosis; Nuclear decondensation; Phagocytosis.

Figure 1. Schematic overview of experimental design for isolation, culture, and differentiation of neutrophils and macrophages

(A) Shown is an illustration of the mouse bone marrow extraction, cell cryopreservation, thawing, recovery and lineage depletion. After thawing the bone marrow, the cells recovered for 24 h in progenitor growth media, then lineage depleted for enrichment of hematopoietic progenitor cells. (B) Depiction of the ex vivo culture process leading from lineage-depleted (Lin⁻) bone marrow cells to terminally differentiated neutrophils and macrophages. The Lin⁻ cells were cultured in lineage growth media with SCF/IL-3 for 3 days to generate progenitors. The derived common myeloid progenitors (D3 CMP) were then divided into two separate cultures, using the recommended cytokine cocktail in lineage growth media for inducing either neutrophil or macrophage differentiation for the indicated number of days.

Figure 2. Visual microscopic analysis of neutrophil vs. macrophage morphology

(A) Representative 60X oil emersion photomicrographs of Wright-Giemsa stained cytocentrifuged cells during the differentiation process revealed changes in nuclear and cytoplasmic features. The progenitors (D3 CMP) exhibit a high N/C ratio (examples indicated with open arrows), whereas neutrophils exhibit characteristic lobulated nuclei (block arrows) as compared to macrophages that exhibit increased cell sizes along with condensed, round nuclei (arrows in D5 pro-MΦ and D10 MΦ). The scale bar in each image indicates 20 μm. (B) Using the cytocentrifuged stained cells from the above imaged slides, the ex vivo cultures were visually examined for morphologic features and categorized into a stage of differentiation for which the percentages were calculated for each timepoint indicated. Shown are graphs of cells identified with features consistent with progenitors (high N/C ratio), myelocyte/monocyte-like (mid-N/C ratio), macrophage-like (low N/C ratio) or neutrophil-like (lobulated nuclei), with average percentages ± standard deviations (SD) indicated from at least three independent inductions of thawed bone marrow cells. Indicated p values were calculated using an unpaired two-sample Student t Test assuming equal variances.

Figure 3. Analyses of immunolabeled cell surface marker protein expression profiles in populations of differentiating neutrophils and macrophages with imaging flow cytometry

(A) Shown are overlaid histograms of intensity within the MC (master channel) mask for each of the corresponding channels, for each antibody fluorophore to evaluate the expression profiles for Gr-1, Mac-1, and F4/80 exhibited by the ex vivo cultured cells induced to either neutrophils (left panels) or macrophages (right panels). The patterns indicate typical changes as neutrophils show increased Gr-1 and Mac-1 expression but predominately lack F4/80, whereas macrophage induced populations exhibit loss of Gr-1 expression and increased Mac-1 and F4/80 expression. (B) Representative compensated images of D3 progenitors (CMPs), differentiating neutrophils (D5, D7 PMN) and macrophages (D5, D7, D10 MΦ) are shown to demonstrate the expression and localization of the three cell surface markers along with DNA staining to illustrate changes in nuclear shape. The scale bar indicates 7 μm, and yellow numbers represent calculated N/C ratio for the event; all images were acquired using the ImageStream instrument at 60X magnification and image display gains for each CSM channel (Ch02, Ch03, Ch11) were set to the same parameters with the IDEAS software to allow for visual comparison of intensity expression between events shown.

Figure 4. Gating and masking strategies with imaging flow cytometry for assessment of nuclear-to-cytoplasmic ratios during neutrophil or macrophage differentiation

(A) All events were initially plotted for the Gradient Raw Mean Squared (RMS) values for the customized brightfield mask (AdaptiveErode(M01, Ch01, 95) vs. the nuclear stained images with the Morphology(M07, Ch07) mask. The initial gating parameter “Focus BF+Nuc” selected events for both the brightfield image and the nuclear DNA stain images that were in-focus (left panel; representative example images shown below demonstrate events that were out of focus (labeled as “a”), compared to the selected in-focus images (labeled as “b”)). The gated FocusBF+Nuc events were then measured using the aspect ratio value compared to the area of the brightfield mask (AdaptiveErode(M01, Ch01, 95)) (center panel, inset image and equation describes the aspect ratio assessment criteria; representative images for “Single BF” events (c) and multiple or aggregated cells (d) are shown below). The gate was set for Single BF events with a high aspect ratio (>0.6) but low area (<600) to exclude doublets or multi-cell aggregates. The Single BF events were compared using the nuclear DNA stain mask (Morphology(M07, Ch07)) for the nuclear area vs. aspect ratio (right panel with images illustrating multiple nuclei (e) and single nucleus events (f)) and gated for “Single BF+Nuc” events. The resulting subpopulation of Single BF+Nuc events was used for the subsequent analysis of cell surface marker expression levels and N/C ratio assessment. (B, C) Examples of customized mask options (masked region displayed in teal overlay) for either brightfield images (B) or nuclear intensity detection (C) are provided, with inserted blue or yellow values representative of the area value for each described mask, illustrating the necessity for careful mask selection that accurately represents the object image, particularly when evaluating subtle changes such as with N/C ratio calculations. Scale bar indicates 7 μm. (D) The N/C ratio value was calculated for the gated “Single BF+Nuc” subpopulation (described above) using a combined feature calculation of the average area from three brightfield masks or three Ch07 masks (calculated in Eq. 1–3, masked selected are labeled with asterisks in B and C). Using this method for evaluating the N/C ratio was determined for the ex vivo cultured cells at specific stages of differentiation (progenitors (D3 CMP), neutrophils (D5 and D7 MN), and macrophages (D5, D7, and D10 MΦ)). The histograms shown depict the distributions of N/C ratios for the indicated cell types, as calculated by the software. Representative images (brightfield, DNA stained, and merged) illustrating the change in N/C ratio as the D3 CMPs undergo differentiation to either neutrophils or macrophages are also shown. N/C ratio value is listed in yellow for each event. Scale bar indicates 7 μm.

Figure 5. Masking and gating strategies for analysis of phagocytosis by imaging flow cytometry

(A) Depicted are three scatter plots with gated areas indicated for each selection strategy, in this case used to analyze neutrophils after phagocytosis of the pHrodo-Green E. coli bioparticles. All events were initially plotted for the Gradient Raw Mean Squared (RMS) values for the customized brightfield mask (AdaptiveErode(M01, Ch01, 95) vs. the nuclear stained images with the Morphology(M07, Ch07) mask. The initial gating parameter “Focus BF+Nuc” selected in-focus events for both the brightfield and the nuclear DNA stained images (left scatter plot). The gated FocusBF+Nuc events were then measured using the aspect ratio value compared to the area of the brightfield mask (AdaptiveErode(M01, Ch01, 95) (left-center panel), and the gate was set for “Single BF” events with a high aspect ratio (>0.6) but low area (<600) to exclude doublets or multi-cell aggregates. The Single BF events were compared using the nuclear DNA stain mask (Morphology(M07, Ch07)) for the nuclear area vs. shape ratio (right-center panel, inset image and equation describes the shape ratio (SR) assessment criteria) and gated for “Single BF+SR_Nuc” events. The resulting subpopulation of Single BF+SR_Nuc events was used for the subsequent analysis of channel 2 (Ch02) fluorescence intensity of the combined mask (MC), and the results were gated to determine the percentage of events positive for pHrodo-Green E. coli bioparticles (pHrodo⁺) or negative for particle engulfment pHrodo⁻, as shown in the representative histogram (right panel). (B) Shown are representative images of nine masks (masked region displayed in white (gray) overlay) that were used to calculate the spot count feature of the corresponding masked region (value indicated in yellow text of each image) on 4 different stimulated neutrophils. The first two masks are the default settings provided by the IDEAS software, which are either not sensitive enough or too sensitive. The evaluation of several masking options lead to optimal mask settings as depicted in the last three images. The final average spot count values (indicated in blue within the left most BF images) were calculated from the three optimal mask settings. The white bar in the lowest set of images indicates 7 μm.

Figure 6. Quantitative measurements of phagocytosis by neutrophils using imaging flow cytometry

(A) Neutrophils generated after 7 days of ex vivo culture of bone marrow progenitors were stimulated with pHrodo-Green E. coli bioparticles while also stained with NucBlue. Live cells were analyzed and images were acquired after either 45 min (at 37°C or 4°C) or for 2 h (at 37°C) of incubation. The percentages of cells positive (pHrodo⁺) for fluorescence particles (spots) were then calculated and reported as percent phagocytosis (left graph). The average number of fluorescence particles (spot count) per event was also calculated and graphed for D5 and D7 PMN stages of differentiation, and indicated as phagocytic index (right graph). Data was generated and graphed from 20X images (D5 and D7) vs. 60X images (D7 only); values inserted above each bar indicate actual average percentages or index values. (B) Distributions of spot counts for each analyzed event were graphed from either 20X or 60X images, with numbers of spots shown as Normalized Frequencies (left two graphs). Also displayed is the graph of the percentage distribution for the number of spots per cell generated by manually counting the 60X images (308 images assessed), of the same D7 PMNs processed by the imaging flow cytometer (right graph). Data shown are representative of at least 2 independent assays. (C) Images of representative cells with green fluorescence (phagocytosed bacteria, E. coli) vs. nuclear staining (red fluorescence, NucBlue) and merged images are shown for cells exhibiting 1, 3 or 15 spots. All images shown were obtained from an ImageStream with a 60X objective. The scale bars in the leftmost brightfield images indicate 7 μm.

Figure 7. Quantitative measurements of phagocytosis by macrophages using imaging flow cytometry

(A) Macrophages generated by 5, 7 or 10 days of ex vivo culture of bone marrow progenitors were stimulated and stained as described for neutrophils, each for 45 min at 37°C or 4°C as a control. Shown are the percentages of phagocytic positive cells (left graph) or phagocytic indices (right graph) at each indicated stage of macrophage differentiation. (B) Distributions of spot counts for cells after 10 days of culture are shown as determined by imaging flow cytometry with 20X or 60X magnification (left two graphs), or using manual counts from the 60X images (right graph). (C) Images from macrophages that have engulfed E. coli particles are shown (1, 5, or 15 spots), including nuclear staining along with merged images; all images were obtained with the ImageStream at 60X magnification and scale bars indicate 7 μm.

Figure 8. Induction of early-stage NETosis in primary neutrophil cultures detected by confocal microscopy

(A) Representative images are shown of untreated vehicle controls (PBS, left column) and stimulated cultures (LPS, PMA, or A23187), incubated for 60 min. Cells were labeled with DAPI (DNA) and FITC-anti-myeloperoxidase (MPO) antibody to visualize MPO expression. Control cell images exhibit the typical lobular nuclear morphology, whereas numerous cells stimulated with LPS or PMA show nuclear decondensation. By comparison, most A23187-stimulated cells exhibit loss of donut-shaped, lobular nuclei. Most stimulated cells exhibited increased MPO expression, some of which was closely associated with nuclear regions stained with DAPI. (B) Images are shown of untreated vehicle controls (PBS, left column), and stimulated cultures incubated with PMA, or A23187 for 60 min, each stained with both DAPI and Sytox-Green. The control cells exhibit typical lobular nuclear morphology, whereas some of the stimulated cells show extracellular regions of DNA stain indicating NET release. All images were taken at 40X magnification, with scale bars (top left panels in A and B) indicating 20 μm. Inserts are shown as 200% zoom settings relative to the parent image.

Figure 9. Gating strategies for identifying cell images for quantitative measurements of NETosis

(A) Gates were applied to the cell populations stimulated with different NETosis inducers to exclude debris and multiple cell aggregates, each indicated by regions within a series of scatterplots. First, events in brightfield views were selected for in-focus cells (Area vs. gradient RMS) to exclude cell doublets and debris. The gated region selected for the next analysis is indicated (left panel). Only those events with spot distances between 0–1 were selected (as indicated with the horizontal line, middle panel), allowing for the selection of cells with either a single nuclear signal or an elongated signal that is attached (e.g. ejecting DNA), but exclude doublets (cells shown below histograms depicting nuclear features are from neutrophils with diffusing or ejecting DNA, or clearly a doublet of two cells). The selected cells were then gated for nuclear fluorescence intensity (Mean Pixel_Morphology, Y-axis) vs nuclear area (Area_Morphology, X-axis) (right panel). The events within the gated region indicated by the large rectangle in the last scatter plot were then further analyzed. (B) The cells produced by the first series of gating strategies then were analyzed for nuclear circularity (nuclear stain, Y-axis, labeled as Circularity_Morphology(M07,Ch07)) vs. cell circularity (brightfield, X-axis, labeled as Circularity_AdaptiveErode(M01,Ch01,95)), and four different quadrants were delineated within the scatterplots for quantitative assessments (lines depict each quadrant). The images shown were obtained from events in each quadrant of the analyzed cells stimulated with each agent, providing representative pictures of cells at each stage of NETosis as identified by each quadrant. (C) The gating strategy was applied to each of the populations of stimulated cells, and shown are the resulting scatter plots with depicted quadrants plus percentages of cells in each quadrant. All plots shown were generated with the data collected with the ImageStream using a 60X objective with cells stimulated for 1 hour with each inducing agent to cause NETosis.

Figure 10. Quantitative imaging of cell vs. nuclear circularity during NETosis identifies four distinct stages

Events from the four quadrants of the scatter plots were quantified and graphed, which indicates the percentages of cells that are at each stage of NETosis. Percentages shown are representative of three independent ex vivo cultures of derived neutrophils stimulated with each agent. Also shown are representative images of the cells stimulated with PMA and A23187 as acquired from the ImageStream, highlighting the changes in both nuclear and cell morphology as identified with DNA staining, brightfield imaging, and expression patterns of MPO, and Mac-1.

Figure 11. Analyses of phagocytosis with E. coli stimulation reveals NETosis by imaging flow cytometry

Images of neutrophils that were stimulated with pHrodo-Green E. coli bioparticles were analyzed for cellular and nuclear circularity using the IDEAS software. (A) Shown are examples of the masking strategy (masked region displayed in teal overlay) used to accurately determine the spot count. The first two sets of images (No mask and M01) are those of the cells with stained nuclei to show examples in the brightfield and the default mask, respectively, indicating that particles outside of the cells are detected (note masking beyond the cell membrane in those under M01). The masking strategy was then adjusted to capture images labeled “Combined Mask 1”, using the mask AdaptiveErode((M01, Ch01, 95), Combined, 8), and those labeled “Combined Mask 2”, using Range(LevelSet(AdaptiveErode(M01, Ch01, 95), Combined, 8), 1 00–5000, 0–1)). The last four images illustrate those used while modifying the masking strategy leading to the combined masks actually utilized. (B) Scatter plots for E. coli and LPS stimulated cells are shown with the quantitatively analyzed quadrants indicated within each plot (left panels), plus the resulting percentages of cells in each quadrant (right graphs). Cell circularity feature indicated in the X-axes utilized different masks for each stimuli, due to alternative masking required to accurately detect cell circularity when analyzing cells with E. coli bioparticles, which are not present in the LPS-stimulated cells. (C) Shown are images of cells representative of those found in each quadrant for NETosis analyses, each showing the numbers of spots identified in the images (yellow text). Each image identifies the fluorescence spots indicating phagocytosis, plus changes in nuclear and cell morphology (e.g. cells undergoing phagoNETosis).