Red blood cell lysis buffer

Healthy adult Chinese donors’ heparinized whole blood (n = 10) was mixed with an equal volume of 3% dextran T-500 (Pharmacosmos, Holbaek, Denmark) in 0.9% NaCl and incubated 20 min at room temperature. Aspirated leukocyte-rich plasma and centrifuged 10 min at 1000 rpm, 5 °C. Discarded supernatant and resuspended cell pellet in a volume of 0.9% NaCl equal to the starting volume of blood. Layered 10 ml Ficoll-Hypaque solution (Solarbio, Beijing, China) beneath the cell suspension which ≤40 ml, and centrifuged 40 min at 1400 rpm, 20 °C with no brake. Aspirated the top layer as well as the Ficoll-Hypaque layer, leaving the neutrophil/RBC pellet, and removed residual RBC with red blood cell lysis buffer (TBDScience, Tianjin, China). After washing with Hanks buffered saline solution (Solarbio, Beijing, China), the cells were resuspended and cultured in IMDM (HyClone/Thermo Fisher Scientific Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS, HyClone), 100 IU/ml of penicillin G and 100 μg/ml streptomycin (Invitrogen, Carlsbad, CA, USA) in a cell incubator (5% CO₂ at 37 °C)46 . The morphological characteristics and purity of neutrophils were examined with Giemsa staining, immunofluorescence staining and flow cytometry as described below.

Neutrophils were isolated from the peripheral blood of healthy cattle by Ficoll-Hypaque gradient centrifugation as previously described [12 (link)]. Briefly, neutrophils were isolated immediately under sterile conditions with Ficoll (TBD Science, Tianjin, China). The mixed red blood cells were then removed with the Red Blood Cell Lysis Buffer (TBD Science), and the cells were resuspended in DMEM medium supplemented with 10% FBS.
A chemotaxis assay was carried out as described previously [20 (link)]. Briefly, neutrophils (1 × 10⁵/mL) were resuspended in 200 μL DMEM medium and added to the upper Transwell chamber (3-μm pores, Costar, Corning, NY, USA). Next, 600 μL culture supernatant from each group of EBL cells infected with M. bovis HB0801, T6.93 (M. bovis ΔMbov_0145), and its complementary strain CT6.93 for 24 h were centrifuged at 10,000× g for 5 min to collect the mycoplasma-free and cell-free supernatant. DMEM medium from the uninfected EBL cells served as the negative control. These culture supernatants were added to the lower wells. The Transwell plate was then incubated at 37 °C for 8 h. A hemocytometer was used to count the cells that migrated to the lower chamber. The Transwell migration assay was repeated three times independently.