Antibody used for FC Figure S1. Multimodal characterization of NIR dyes in vitro Figure S2. Ex vivo analysis of HL60 cells homing

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1 Antibody used for FC The following antibodies were used following manufacturer s instructions: anti-human CD4 (clone HI3, IgG1, k - Becton Dickinson), anti-human CD33 (clone WM3, IgG1, k- Becton Dickinson), anti mouse CD4 (clone 3-F11 - RatIgG2b, k- Becton Dickinson), anti-mouse CD11c (clone S- Becton Dickinson), anti-mouse Sca-1 (clone D7, Rat IgG2a, k ebioscience), anti- mouse CD117 (clone 2B8, RatIgG2b, k Becton Dickinson) and anti-mouse lineage markers (Lin) (Stem Sep, Mouse hematopoietic progenitor cells enrichment kit StemCell Technologies). Figure S1. Multimodal characterization of NIR dyes in vitro HL6 cells were stained with DDAO-SE (DDAO),, or IRDye 8 CW (IRDw8) using 1.2-to -µm final dye concentrations as specified on the figure. Quantum-dot Q-tracker 7 was evaluated using either the standard protocol (1 ),. or 2 times (. and 2, respectively) of the recommended nano-particles dose. After washing, overnight incubation in standard culture conditions at 37 C, cells were washed again before being plated together with approximately 3% HL6-eGFP + sorted unstained cells. Individual wells were separately analyzed at day (D), day 4 (D4) and day 6 or 7 (D6/7) using the same settings on a NIR scanner and flow cytometry. Labeled cells were also imaged by confocal microscopy. (A) Staining efficiency and behavior of NIR fluorescence intensity depending on time: Cell suspensions were collected and analyzed by flow cytometry. The specific mean fluorescence intensity (smfi) was calculated by doing the ratio of the geometric mean fluorescence intensity of NIR positive cells over NIR negative cells used as control. Unmanipulated plates were also analyzed using the Odyssey scanner (Ai). DDAO was analyzed using the 7 nm channel (red); and IRD8 fluorescence were imaged using the 8 nm channel (green). Integrated fluorescence intensity (AU = arbitrary unit) was quantified for each well. The diagram (lower panel) represents the mean value of 2 wells (Aii). (B) Assessment of dyes / staining procedure-related cytotoxicity. The relative proliferation capacities of egfp - NIR-stained cells was compared within the same well to the proliferation of egfp + unstained cells for each time point by flow cytometry. (C) Confocal analysis of stained cells: In a distinct experiment, HL6 cells were stained using µm of each dye and imaged in-vitro by confocal or multiphoton (Q-tracker) microscopy. Figure S2. Ex vivo analysis of HL6 cells homing Main hematopoietic organs as well as the kidney have been dissected from day 1 animals (Fig. 1) and imaged. Fluorescence intensity is displayed on the right size of the images. Note: a very strong NIR signal emanating from the spleen as well as the tail from the left animal is saturating the PMT (white pixels) despite using the lowest level of sensitivity of the scanner. Abbreviations: Sk: skull; Sc: scapula; Kd: kidney; Sp: spleen; H: hip; F: femur; T: tibia; Tl: tail. Figure S3. Lipophilic dyes but not CFSE induce MEC in the absence of phagocytosis Top lane: staining efficiency of HL6 cells (after washing) used for the co-culture from Fig. 3 and used for the direct co-culture without a culture period (HL6 before co-culture). Gates in colour represent the positive threshold established on unstained HL6 cells. Bottom lane: Fluorescence intensity associated with stromal cells (CD4 ) co-incubated with the above HL6 cells for overnight incubation. Coloured gates represent the positive threshold established on unstained stromal cells.

2 Figure S4. Kinetic of MEC after 1 and 24 hours of co-culture HL6 cells or HL6 cells expressing high levels of GFP (GFP) were stained in PBS without serum with CFSE (.µm) 31, DiI, PKH26 or DiD (2µM) for mn and washed twice in PBS 2% FCS. Cells were then incubated 24h in standard culture conditions before being plated on fibroblasts. After 1 h or 24 h of co-culture at 37 C, cells were recovered by trypsinization, stained for hcd4 + hcd33 (both PE in the case of DiD, APC for all other dyes) and analyzed by flow cytometry. The absolute amount of fluorescence found associated with the fibrolasts is displayed as a percentage of the total fluorescence for each dye (average of 3 to 4 wells). NS: Non Significant. Figure S. Unsuccessful strategies to abrogate MEC (A) HL6 cells (left panel) where stained with µm in standard conditions (mn at 37 C) in PBS without serum (central panel) or in serum free medium (SFM) StemSpan (right panel) diluted ½ in PBS without serum. (B) HL6-GFP bright cells stained with µm DiD (upper central panel). They were washed 2, 3 or 4 times in PBS 2% FCS or sorted by flow cytometry on the live (DAPI negative) DiD + criteria. These cells were then co-cultivated on a confluent stroma-layer for h and the transfer toward stromal cells (GFP negative) was analyzed by FC. Displayed are stromal cells DiD fluorescence. Gates represent the threshold of positive events based on analysis of unstained GP293 cells (top left). Identical experiment were conducted in parallel with DiI and leading to similar results (cf: below). (C) Absolute quantification of dyes distribution between HL 6 cells and stromal cells in the same experiment for each one of the dyes (average viability = 7% ranging from 6% for DiD FACS to 78%). Figure S6. Flow cytometry and bioluminescence-based optimization of staining procedures for murine HSPCs Bone marrow cells from Beta-actin luciferase mice were recovered, lineage depleted and stained mn at 37 C in PBS with DiI, and PKH26 using indicated concentrations. Unprocessed cells were also kept as control (UP). After 2 washings, cells were plated in SFM supplemented with cytokines. After 24h culture, some cells were recovered, washed and suspended in DAPI containing PBS 2% FCS for FC analysis. (A) Viability (DAPI negative) of the cells after 24h culture (representative of 2 independent experiments). (B) Specific geometric mean fluorescence intensity (sgmfi) of the cells after 24 hours (cf: Fig. S1A). (C) Some cells had also been plated in triplicate in similar conditions in 96 well plates under µl for evaluation of their functional capability to proliferate. 7 days after seeding, luciferin was added to the wells and bioluminescent (BLI) activity of each well was quantified over a 2mn period of time. BLI activity is expressed as a percentage of the respective negative control (since PKH26 staining has to be done in the buffer provided in the kit and DiI and staining are performed in PBS).

3 Figure S1 A (i) Flow cytometry (ii) NIR scanner D D4 D7 1.x 1x 2x sgmfi 1.2 Q-7 DDAO IRDw8 B % of GFP- cells DDAO IRDw8 D D4 D7 Fluo. Intensity (AU) 4 3 Medium Cells 1.2 DDA Medium D D4 D6/7 Cells 1.2 Medium Cells 1.2 IRDcw8 C

4 Figure S2 HL6- Control

5 Figure S3 HL6 cells 99. % 67.2% 99.9 % 89.2 % before coculture CFSE DiI PKH26 GP % % 8.6 % 41.6 %

6 Figure S4 % of absolute signal h 24h NS NS p=.21 NS NS GFP CFSE DiI PKH26 DiD

7 Figure S HL6 staining in PBS staining in SFM A SSC-A B GP293 FACs sort SSC-A 2 washes 3 washes 4 washes HL6 DiD stromal C % of total stained cells w 3w 4w FACs 2w 3w 4w FACs 2w 3w 4w FACs DiI DiD

8 Figure S6 A B % viable cells sgmfi 1 DiI PKH UP DiI PKH C BLI activity (% of unstained) 2 1