Easysep mouse pe positive selection kit 2

Murine AMs were isolated from BALF as described previously (17 (link)). A total of 7 ×10⁶ AMs were obtained from 20 mice. The AMs were surface-stained with an anti-mouse PE-CD64 antibody (139304, BioLegend, CA, USA) and then positively selected and enriched with an EasySep™ Mouse PE Positive Selection Kit II (17666, STEMCELL Technologies, Vancouver, Canada). CD64+ AMs were grown at a density of 4 ×10⁵ cells/well in a 24-well plate using RPMI 1640 culture medium (Thermo Fisher, Chengdu, China) containing 10% FBS (Life Technologies, MA, USA) and were challenged with influenza H1N1 virus (multiplicity of infection, MOI=3). In some of the AM stimulation groups, recombinant proteins of the chemokines CXCL1 (453-KC), CXCL2 (452-M2), and CXCL5 (433-MC) (R&D Systems, MN, USA) were added to the culture medium (30 ng/ml each). The CXCR2 antagonist SB225002 from Selleck Chemicals (TX, USA) was used at 60 nM in the CXCR2 antagonism experiment. The PI3K inhibitor LY294002 (L9908, Sigma-Aldrich, MD, USA) and MEK inhibitor PD98059 (P215, Sigma-Aldrich, MD, USA) were used at 25 μM. After 10-20 h of incubation, the cells were harvested for detection of chemokine expression by reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay, and the culture supernatants were collected for detection of chemokine protein levels by ELISA.

To ablate endogenous BM, WT B6 mice were irradiated to 9 Gy using an X-RAD 320 (Precision X-Ray). After a 6-hour rest period, mice were transplanted with 4 × 10⁶ donor BM cells via i.v. tail vein injection. BM cells were flushed from the femurs of donor WT B6 or IL-12Rb2–KO mice. Marrow was subjected to RBC lysis (Thermo Fisher Scientific) and filtered (30 μM). For BM mixing studies, untouched WT BM CD3⁺ cells were isolated from WT B6 donor marrow with the EasySep Mouse T cell Isolation Kit (Stemcell) from Stemcell used per manufacturer recommendations. Untouched IL-12Rb2–KO CD3^– cells were isolated from IL-12Rb2–KO donor marrow by using positive selection to remove CD3⁺ cells with a PE-conjugated CD3 primary antibody (BioLegend), followed by positive selection of PE-labeled cells with the EasySep Mouse PE Positive Selection Kit II (Stemcell Technologies) used per manufacturer recommendations. Transplanted mixed BM consisted of 10% CD3⁺ and 90% CD3^– cells. Mice were allowed 3 weeks to engraft new marrow before being transplanted with MOC22 cells and used to in vivo studies.

Peripheral lymph nodes including a total of six axillar, cervical, and inguinal lymph nodes and spleen from each mouse were pooled for making single-cell suspensions using slides to smash tissues in 10 ml of HBSS supplemented with 3% FBS. RBCs were lysed with ACK (ammonium-chloride-potassium) lysis buffer (Lonza) followed by filtration with a 70-μm cell strainer. The cells were subsequently incubated with PE-conjugated Abs against CD43 and CD9, followed by purification using an EasySep mouse PE positive selection kit II according to the manufacturer’s instruction (STEMCELL Technologies). The purity of follicular (FO) B cells was ∼95%. All downstream experiments were performed using purified FO B cells.