Ficoll

For PBMC isolation, the whole blood was diluted with an equal volume PBS before performing the centrifugation steps. Then, the blood was carefully added to the Ficoll (Tbdscience) layer (blood:Ficoll = 1:1) and centrifuged at 550g for 20 min with the brake off. The cellular layer at the Ficoll/PBS interface was aspirated and washed twice with PBS, removing the residual Ficoll. Approximately 1 × 10⁶ of PBMCs could be isolated from 1 mL of peripheral blood.

Considering that autoreactive PBMCs, mainly lymphocytes, may participate in the autoimmune inflammatory process, we chose PBMCs as a source for determining MLKL mRNA level in SLE patients. The venous blood samples (4–5 mL) were collected in an EDTA-K2 tube from all the participants before breakfast, and PBMCs separated within 2 h by Ficoll (TBD Science, Tianjin, China) gradient centrifugation for 30 min at 1700 r/min. PBMCs were then transferred into 1 mL TRIzol Reagent in 1.5 mL centrifuge tubes and stored at − 80 °C until RNA extraction.
Total RNA was extracted from PBMCs by using TRIzol Reagent (Invitrogen, CA, USA) according to the manufacturer’s protocol and quantified with the NanoDrop ND-1000 (Thermo Scientific, USA). Approximately 200-800 ng of RNA was obtainted from 1mL of venous blood samples. Samples were used only if the ratio of the absorbance at 260 nm to that at 280 nm (A 260/A 280) was between 1.8 and 2.1. RNA samples with concentrations > 0.2 μg/μL were used for following reverse transcription reaction.

Mononuclear cells were isolated from umbilical cord blood (The First Affiliated Hospital of the University of Science and Technology of China) and prepared from buffy coats obtained from healthy infant donors by centrifugation and a Ficoll system. Cord blood was layered on top of Ficoll (tbd science, # LTS1077) in a 50-mL tube and centrifuged at 500 x g for 30 min at room temperature. Buffy coats were collected, washed twice in phosphate-buffered saline (PBS), and finally resuspended. Peripheral blood stem cells were collected from the peripheral blood through a process known as apheresis. Donors were given daily subcutaneous injections of G-CSF, to help mobilize stem cells from the bone marrow and into the peripheral circulation.

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.

The leukemic bone marrow or peripheral blood samples (leukemia blast cells >30%) from patients with AML were collected at the time of diagnosis or relapse in the West China Hospital, Sichuan University (China) from 2015 to 2017, and blood samples from healthy subjects were used as control. Written informed consent was obtained from the patients, and the study was approved by the local ethics committee. Mononuclear cells were isolated by Ficoll (TBD Science, Tianjin, China) density gradient centrifugation.

The staff of the sample bank will standardize and collect the follicular fluid produced in the process of assisted reproductive technology treatment of the included patients. At the first puncture of each ovary from which the egg was retrieved, the follicular fluid samples were collected from a single large follicle and centrifuged to remove cells for subsequent analysis. The tops of cell precipitates were collected from concomitant follicular fluid samples of PCOS patients, which were aspirated and washed in PBS. Then, the cell precipitates were resuspended in PBS and cultured in Ficoll (LTS1077, TBD Science) to layer the solution and separate from red blood cells through centrifugation. The cell layer at the Ficoll/PBS interface were aspirated and washed with PBS to remove residual Ficoll. The final granulosa cells were precipitated in PMI-1640 medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin (5000 U/mL) at 37°C in a moist atmosphere of 5% CO₂ and used for subsequent assays.