Cd11b fitc

Rats were perfused with PBS, and total brain was extracted. Brains were enzymatically and mechanically dissociated into a cell suspension using the Adult Brain Dissociation Kit and gentleMACS Octo Dissociator (Miltenyi). Microglia were isolated using CD11b/c magnetic microbeads (Miltenyi) according to the manufacturer’s instructions. Microglia were used immediately for RNA and protein extraction. For validation of microglia purity, microglia were also plated in microglia media (1X MEM, 4% Fetal Bovine Serum, 6% Horse Serum, 0.6% glucose, 1 mM sodium pyruvate, 1mM L-glutamine, and 1% pen/strep) in 37 °C and 5% CO2. Purity was confirmed with FACS analysis of CD11b and CD45 expression with CD11b-FITC and CD45-APC-Vio770 respectively (Miltenyi). Data supporting the purity of the microglia isolation are contained in the Supplemental Fig. S2.

On day 1 after ICH, mice were anesthetized with 3% isoflurane and were perfused with cold phosphate-buffered saline. Brains were cut into 1-mm-thick coronal sections with a mouse brain slicer matrix (Zivic Instruments, Pittsburgh, PA). Four 1-mm-thick brain sections of the left striatum with hemotoma were collected. The striatum was dissociated with GentleMACS Dissociator (Miltenyi Biotec, Auburn, CA) as described previously^{28 (link),30 (link)}. The final cell suspension were incubated with the primary antibodies: CD11b-FITC (Miltenyi Biotec, 130–113-234) and ACSA-2-APC (Miltenyi Biotec, 130–117-535) or and Ly6G-APC (Pharmingen, 560,599) for 30 min at 4ºC. The corresponding isotype antibodies were used as negative control. Propidium iodide (Sigma, St. Louis, MO) staining was used to exclude dead cells. Cell supernatants were analyzed by CytoFLEX cytometer (Beckman Coulter, Indianapolis, IN) with CytExpert software 2.0 (Beckman Coulter). Macrophage/microglia cells were distinguished as CD11b-FITC⁺ / ACSA-2-APC^- population. Astrocytes were distinguished as CD11b-FITC^- / ACSA-2-APC⁺ population. Neutrophils were distinguished as CD11b-FITC⁺ / Ly6G-APC⁺ population. Gated cells were collected into TRIzol reagent (Qiagen, 217,004) for mRNA extraction and real-time PCR.

Mice of generation x + 1 with signs of emerging hind limb paralysis or elevated CD19⁺ blast levels in the peripheral blood were anesthetized with ketamine/xylazine, and the animal was exsanguinated via the retrobulbar venous plexus. After cervical dislocation for confirmation of death, spleen and femoral bones were dissected and collected in cold PBS. Bone marrow and spleen cells were isolated as described before (Richter et al. 2020 (link)). For flow cytometric characterization of the blast population using FACSVerse (Beckton Dickinson) and FACSuite software, the following antibodies were used: CD3e-FITC, CD11b-FITC, CD8a-PE (all Miltenyi Biotec), CD34-FITC, CD45R-FITC, CD3e-PE, Sca-1-PE, CD4-APC, c-kit-APC, CD45-PerCP-Cy5.5 (all BD), CD19-PE, IgM-APC (all Biolegend). Doubling times of leukemic cell populations were calculated from peripheral blood blast frequencies using the following formula with t₁ and t₂ indicating the age of the animal in days at the respective time points of sample analysis: $$\mathrm{Doubling}\mathrm{time}=\frac{\left(t_2,-,t_1\right)\times \log (2)}{\log (\mathrm{blast}\mathrm{frequency}{(t}_2))-\log (\mathrm{blast}\mathrm{frequency}(t_1))}$$
Cytospins were prepared from spleen and bone marrow cell suspensions and stained as described before (Richter et al. 2019 (link)). For monitoring of CD19⁺ blast frequencies, blood (< 50 µl) was sampled from the tail vein, and CD19⁺, c-kit⁺ and Sca-1⁺ cell populations were quantified as described above.

The primary brown adipocytes were prepared in accordance methods with the previous publication [20 (link)]. Floating adipocytes were separated from the SVF (Stromal Vascular Fraction) by centrifugation at 300 ×g for 3 min. SVF was sequentially filtered through 70 μm filters before staining with the following antibodies for 10 min on ice: Sca-1-APC (Miltenyi Biotec, 130-093-223), CD11b-FITC (Miltenyi Biotec, 130-081-201), and CD45-PE (Miltenyi Biotec, 130-091-610). Following antibody incubation, cells were washed, centrifuged at 300 ×g for 10 min, and sorted with a BD FACS Aria (BD Biosciences, CA, USA). Data analysis was performed using BD FACS Diva software.

Mice (n=7+7 in both CUS and microinjection) were sacrificed with CO₂. Tissues were gently homogenized through 70µm cell strainers (#352350, BD Biosciences) on ice as we described previously (24 (link), 32 (link)). Homogenates were blocked in PBS+10% rat serum for 1h with gentle rotation at 4°C. Fluorescent antibody makers (0.5µl/marker, mostly from Biolegend) diluted in 200µl PBS+1%FBS were added and incubated for 1h at 4°C with light protection. For CUS experiment, Plxnb2-PE (#145903), CD11b-BV421 (#101251), CD45-BV650 (#103151), and Glast-APC (#130-123-555) were used. For microinjection experiment, CD11b-FITC (#101206), CD45-PE/Cy7 (#103114), Glast-PE (#130-118-344, Miltenyi), and MHCII-BV711 (#107643) were used. Washed samples were finally resuspended with FC buffer and filtered through 35μm cell strainers into flow tubes (#08-771-23, BD Biosciences). The acquisition was made with BD LSR Fortessa™ (BD Biosciences). Data were analyzed using Kaluza (Beckman Coulter). Percentages (%) of positively stained cells were calculated.