Facschorus software

For flow cytometric analysis, cells were harvested in the following manner: Nonadherent cells were first removed by pipette resuspension and rinsed with PBS, followed by the detachment of adherent cells using EDTA (Invitrogen, Carlsbad, USA) (10 mM at 37°C for 5 min) after which an equal volume of PBS was added. This was followed by pipette resuspending to lift adherent cells. Cells were then rinsed with washing buffer (2% FCS, 2 mM EDTA, 0.05% sodium azide, pH 7.4) and centrifuged at 300 g for 5 min. After supernatant removal, nonspecific binding sites were blocked using 10% human serum (diluted in washing buffer) for 20 min at RT. Cells were then washed with 1 mL washing buffer, centrifuged (300 g for 5 min), and the supernatant was removed before the labelling of immune cell populations using the following antibodies in distinct panels (incubated for 30 min on ice and protected from light): CD14-PerCP Cy5.5, CD16-PE-Cy5, CD163-PE-Dazzle594, CD3-BV421, CD86-Alexa Fluor 488, HLA-DR-PE-Cy7, and PD-1-Alexa Fluor 488 (all from Biolegend, London, UK). Cells were immediately analyzed using a BD FACSMelody instrument (BD Bioscience, Heidelberg, Germany) with FACS Chorus software (BD Biosciences). Data were analyzed using FlowJo software v10 (FlowJo LLC, Ashland, USA) according to the gating strategy in Supplementary Data 1.

FACS (fluorescence-activated cell sorting) was performed on lymphoblastoid cell lines to retrieve a homogenous subpopulation of CD123⁺ CD19⁺ cells. Cells were counted using a Neubauer Chamber, washed with DPBS and incubated in blocking buffer (DPBS with 5% (v/v) FBS) for 15 min before the cells were diluted to a concentration of 1 × 10⁷ cells/mL. Subsequently, 2 × 10⁷ cells were labelled with 17.5 µL α-CD123-PE-Cy^TM7 (BD Bioscience, #560,826) and 80 µL α-CD19-FITC (BD Bioscience, #555,412) antibodies for 45 min at 4 °C on an end-over-end rotator in the dark. Cells were washed with FACS buffer (DPBS with 0.1% (v/v) FBS) once and passed through a 40 μm nylon cell strainer (Corning Inc., #431,750) into FACS tubes. Unstained and single-colour control samples were prepared likewise without or single addition of antibodies respectively. Cells were sorted with the FACSMelody™ Cell Sorter (BD Bioscience) using the FACSChorus™ Software (BD Bioscience). Gating for live cells (P1) was done using FSC-A/SSC-A and single cells of P1 were identified with SSC-W/SSC-H (P2) and FSC-W/FSC-H (P3). Cells of P3 double positive for CD123 and CD19 were then sorted. The sorted samples were further analysed in parallel with the bulk sample pellet using dPCR. FACS evaluation analysis was performed using FlowJo™ Software (BD Biosciences).

Cryovials of AML cells were thawed as follows: while 9 ml of Fetal Bovine Serum (FBS) was allowed to come to ~24 °C, AML cryovials were removed from liquid nitrogen, and warmed in a 37 °C water bath until the cells began to thaw. After 1 min, 1 ml of room temperature FBS was added to the warming cryovial with a P1000 pipet tip and allowed to mix with thawing cells. The freshly added FBS was removed from the cell pellet and transferred back to the FBS stock. This process was repeated 3–4 times until all cells from the cryovial could be poured directly into the FBS stock. The empty cryovial was rinsed once more with the FBS mixture. Cells were then pelleted by centrifugation at 300G for 5 min and resuspended in Phosphate-buffered saline (PBS) at a concentration of 1 × 10⁶ cell/ml in 1x PBS. Cells were then pipetted through a 70-µm filter into a 5-ml tube for sorting. Cells were then stained with 1 µl 7-AAD per 1 ml of cells for 30 min at 4 °C. If cell viability was ≤85%, stained cells were filtered through a 40-µM Flowmi cell strainer (Miltenyi), flow sorted, and gated using the FACS Chorus software (BD Biosciences).

Mouse brains were dissociated to cells using the Adult Brain Dissociation Kit (Miltenyi Biotec) following the manufacturer’s protocol with some modifications. Briefly, a fresh whole brain was washed with cold PBS and dissected into small pieces. Aliquots were transferred into C Tubes (Miltenyi Biotec) and mixed with the provided enzymes. C Tubes were attached to the gentleMACS Dissociator (Miltenyi Biotec), and the following gentleMACS programs were performed: m_brain_01_01, m_brain_02_01, m_brain_03_01. Each program was repeated twice with 5-min intervals on a tube rotator at 37 °C. The dissociated brain was applied onto a 70 μm cell strainer and debris and red cell removal steps were performed in accordance with the manufacturer’s protocol.
The separated cells were treated with 1% bovine serum albumin (BSA) in PBS for 30 min, followed by immunostaining with APC-anti-mouse ACSA2 antibody (130-116-245; Miltenyi Biotec, 1:50) and Alexa Fluor 488-anti-mouse CD11b antibody (53-0112-82; Invitrogen, 1:600) for 30 min at 4 °C in the dark. After three-times washes with 0.1% BSA in PBS, cells were separated by BD FACSMelody Cell Sorter along with BD FACSChorus software (BD Biosciences).

Autofluorescence of microalgae enabled the differentiation of M. salina and T. lutea in co-culture. Determination of individual cell counts was conducted using a flow cytometer (BD FACS Melody Cell Sorter; BD Biosciences, San Jose, CA, USA). Analysis was performed with BD FACSChorus software (BD Bioscience). The event rate was set to 1000 events per second. Cell samples were excited by a red laser at 640 nm and the emission was measured by applying the 660/10 nm bandpass filter. Hereby, the fluorescence intensity of the microalgae strain T. lutea was higher, which allowed the separation of the two algae strains (Fig. 2). Fluorescence signals were plotted as histograms with cell counts vs. fluorescence intensity in bi-exponential display. Specific linear correlation factors between cell counts and OD₇₅₀ were determined prior to co-culture experiments by examining the cell counts in manually mixed samples with different ratio combinations of both strains characterized by their OD₇₅₀. All samples were diluted with 25 g L⁻¹ NaCl to obtain an OD₇₅₀ of 0.2.Fig. 2

Histograms of three samples with A 100% M. salina, B 100% T. lutea and C mixed sample containing both algal strains at an OD₇₅₀ of 0.2. X-axis shows the intensity of the fluorescence signal excited by a red laser at 640 nm and detected using the 660/10 nm bandpass filter. Y-axis shows the cell counts of the algae strains