Easysep mouse cd11b positive selection kit 2

Peripheral blood from T127 carrying FVB mice was collected through orbital sinus of isoflurane anesthetized mice. Blood was collected with EDTA blood collection microtubes. After serum aspiration, blood was diluted with PBS and put through density centrifugation with Ficoll. After carefully extracting the layer of the mononuclear cells, cells were washed three times with PBS. CD11b positive cells were isolated by EasySep Mouse CD11b positive selection Kit II (STEMCELL, 18970). The CD11b positive cell pool was incubated with primary C5aR1 antibody at 3 μg/ml in PBS with 2% FBS. Secondary antibodies conjugated with Biotin were added 1.2 μg/ml in PBS with 2% FBS. Magnetic column based cell sorting was performed by following manufacture’s manual of EasySep Biotin Positive Selection Kit II (STEMCELL, 17683). The purity of positive cells was assessed with flow cytometry. 5*10⁵ C5aR1^hi cells in 100 μl selection buffer or only 100 μl selection buffer was transferred to FVB mouse 2 weeks after T22 transplantation through tail vein injection. Olaparib treatment started on the second day after C5aR1^hi cell transplantation with the same dose indicated above.

Mouse BM myeloid cells were isolated according to the protocol, as we have done previously (33 (link)). First, to obtain BM cells, the femur and tibia were flushed using a syringe equipped with a 23-gauge needle, and the resulting cell suspension was collected in the appropriate medium. The suspension was then gently passed through the syringe several times to disperse any clumps. A 70 μm mesh nylon strainer was used to eliminate any remaining clumps or debris from the cell suspension. After resuspension, myeloid cells were isolated using EasySep Mouse CD11b Positive Selection Kit II (StemCell Technologies, 18970), according to the manufacturer’s instructions.

The isolation of microglia was performed as previously described [17 ]. Briefly, mice were anesthetized with a combination of ketamine (0.12 mg/g, i.p.) and xylazine (0.01 mg/g, i.p.), and brains were placed in ice-cold phosphate-buffered saline (PBS) as quickly as possible. Brains were carefully minced into small fragments and digested with trypsin at 37 °C for 20 min. The digested tissue was then triturated and centrifuged at 400 g for 5 min. Centrifuged cells were collected and resuspended in 37% Percoll. The resuspended tissue was placed into 30% Percoll and 70% Percoll was added as the top layer. The tube was then centrifuged once more (700g) for 10 min. The cells between the 30 and 70% Percoll were collected and resuspended in Dulbecco’s Phosphate-Buffered Saline (DPBS)(~2 × 10⁷/mL). Cd11b⁺-positive magnetic isolation was then performed with a STEMCELL Easysep isolation kit (18970, EasySep™ Mouse CD11b Positive Selection Kit II, STEMCELL, Canada). Isolated cells were lysed in Radio Immunoprecipitation Assay (RIPA) buffer and subjected to western blot analysis.

The isolation of microglia was performed as previously described (14 (link)). Briefly, mice were anesthetized, and brains were collected and placed in ice-cold PBS as quickly as possible. Brains were carefully minced into small fragments, and the digestion of the brain was performed at 37°C for 10 min with papain (3 mg/mL) and DNase I. The digested tissue was centrifuged, and the cells were collected and resuspended in 37% Percoll. The resuspended cells were placed in 30% Percoll, and then 70% Percoll was added as the upper layer. The tubes were centrifuged at 3,000 rpm for 20 min without interruption. Cells were collected between the 30 and 37% Percoll and resuspended in Dulbecco phosphate-buffered saline PBS (~2 × 10⁷/mL). CD11b⁺ positive magnetic isolation was performed with a STEMCELL EasySep isolation kit (18,970, EasySep™ Mouse CD11b Positive Selection Kit II; STEMCELL, Canada). Isolated cells were lysed in radioimmunoprecipitation assay buffer (RIPA) and subjected to Western blot analysis.

Bone marrow derived macrophages (BMDM) were derived from isolated monocytes from the marrow of C57BL/6 mice using EasySep Mouse CD11b Positive Selection Kit II (Stemcell Technologies). Alternatively, human monocytes were isolated from discarded de-identified blood donation leukopaks from the Children’s Hospital Colorado Blood Donor Center, after written informed consent in accordance with the Declaration of Helsinki. Leukopak peripheral blood mononuclear cells (PBMCs), obtained using Lymphoprep (Stemcell Technologies), underwent CD14⁺ isolation using MojoSort™ Human CD14 selection kit (Biolegend). Monocytes were matured to macrophages on non-TC treated plates for three to seven days in cRPMI supplemented with 25ng/mL GM-CSF and 5ng/mL M-CSF, after which non-adherent cells were discarded. PtdSer expression on AML cell lines was induced via UV light exposure (15 minutes), followed by incubation at 37° for two to four hours, and then opsonized with 250nM recombinant mGAS6 (R&D Systems). Macrophages were pretreated with 300nM MRX2843 or vehicle for one hour in 24 well plates before AML cells were added (1.0×10⁶ cells/well). After 48 hours, non-adherent AML cells in the supernatant were removed; macrophages were washed with PBS thrice to remove residual AML cells.