VCAM1 and MHCII staining on hematopoietic stem and progenitor cell (HSPC) populations is predominantly remnant associated. Imaging flow cytometry analysis was performed on HSPC populations in Kit-enriched bone marrow (BM) from C57BL/6J mice as described,¹⁷ except that the anti-F4/80 antibody was replaced with the anti-VCAM1 or anti-MHCII antibody, as indicated. (A) VCAM1 was detected on most cells in all HSPC subsets: Lin^–Sca1⁺Kit⁺CD48^–CD150⁺ HSCs, Lin^–Sca1⁺Kit⁺CD48^–CD150^– multipotent progenitors (MPPs), Lin^–Sca1⁺Kit⁺CD48⁺ hematopoietic progenitor cells (HPCs), and Lin^–Sca1^–Kit⁺ committed progenitors (CPs). (B) Representative images of HSCs display cell-surface staining of population-defining markers (Sca1, Kit, and CD150), contrasting with punctate, remnant-associated VCAM1 staining. Violin plots of stain area for each marker (n = 600 cells) demonstrate that VCAM1 stain area was consistently lower than that of Sca1, Kit, and CD150. (C) Manual classification reveals the VCAM1 staining pattern on HSCs, MPPs, and HPCs to be exclusively remnant associated (>300 cells classified per subset), whereas a small fraction of VCAM1⁺ CPs display cell-surface VCAM1 expression. For each classification, representative images of VCAM1⁺ CP are displayed as a single-color fluorescent image and a brightfield overlay with the same image. (D-F) (D) MHCII were detected on most HSCs, MPPs, and HSCs and at a lower frequency on CPs. (E) Manual classification of MHCII staining patterns on HSPC subsets (>500 cells classified/subset), with representative images of MHCII⁺ HSC for each classification. (F) Imaging flow cytometry of BM single-cell suspension generated by collagenase type IV/DNase I digestion (37°C for 40 minutes) of an extruded tibial BM plug, followed by gentle mechanical disruption with a pipette, and assessed for CD11b, F4/80, and VCAM1 expression. (Left) Scatter plot displaying F4/80 and VCAM1 staining intensity for all focused singlets. The pink gate indicates double-positive events, and cells manually classified as having surface staining for both F4/80 and VCAM1 (less than 3%) are highlighted in yellow on the scatter plot. The middle panel shows representative images for abundant remnant F4/80⁺VCAM1⁺ staining patterns, with the left panel showing represented images of the infrequent cells exhibiting surface F4/80⁺VCAM1⁺ staining pattern. These data are representative of the outcome of ex vivo digestion strategies tested on both the BM and spleen, with no substantive gains made in intact macrophage recovery from these hematopoietic tissues. Scale bar, 10 μm. BF, Bright Field.