The study was approved by the Institutional Research Ethic Board at Soochow University. Table S1 showed the basic characteristics of 43 study subjects. The human subjects included 43 unrelated Chinese Han adult females from Suzhou city of China, which were recruited originally for identifying risk molecules of rheumatoid arthritis. Subjects were excluded from serious diseases involving vital organs (brain, liver, kidney, heart or lung). All subjects signed informed consent forms before entering this project. A total of 15 ml peripheral blood was collected and stored in sodium‐citrate‐supplemented vacuum tubes. PBMCs were isolated using density gradient centrifugation using Lymphoprep (Sigma, life science, USA). PBMCs were divided into two equal parts, one for DNA extraction, and the other for RNA extraction after treatment of Trizol reagent (Invitrogen, Carlsbad, CA) to avoid RNA degradation.
Lymphoprep
Manufactured by Merck Group
Sourced in United States, United Kingdom
Lymphoprep is a density gradient medium used for the isolation of mononuclear cells, such as lymphocytes, from whole blood or bone marrow samples. It facilitates the separation of these cells from other blood components by centrifugation.
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Lab products found in correlation
Penicillin streptomycin l glutamine, by Thermo Fisher Scientific (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Six well plate, by Corning (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Ficoll 1, by Merck Group (1 mentions)
Ficoll hypaque, by Merck Group (1 mentions)
Rpmi 1640, by Sartorius (1 mentions)
L glutamine, by Sartorius (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Lymphoprep, by STEMCELL (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Cd62l apc, by BioLegend (1 mentions)
Cd3 apc cy7, by BioLegend (1 mentions)
Cd45ra v450, by BD (1 mentions)
Cd95 bv711, by BD (1 mentions)
7 aad, by BD (1 mentions)
Diva software, by BD (1 mentions)
22 protocols using lymphoprep
1
Isolating PBMCs from Chinese Adults
2
Proteomic Analysis of Immunized Peripheral Blood
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Thirty-two individuals (16 for TFP1 injection and 16 for GVHSV injection) were immunized as described above. Peripheral blood was extracted at 14 dpi, and PB-RBCs were purified by 2 density gradient centrifugations (1600 rpm, Ficoll 1.007; Lymphoprep, Reactiva, Sigma-Aldrich) as previously described [9 (link)]. The 99.9% purity of RBCs was estimated by optical microscopy (Supplementary Figure S1 ). Cells (107 cells per individual) were pelletized by centrifugation (1600 rpm, 5 min), the supernatant was removed, and the cell pellet was washed 3 times with PBS. The pellet was then digested, cleaned-up/desalted, and pooled into 2 pools of 8 individuals for each condition (TFP1 or GVHSV) (Figure 1 ). Samples were subjected to liquid chromatography and mass spectrometry analysis (LC-MS) as previously described [14 (link),29 (link)].
3
Isolation of Mouse Splenic Lymphocytes
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Two weeks after the booster immunization, all immunized mice were euthanized with an overdose of pentobarbital (60 mg/kg) administered intraperitoneally, and splenic lymphocytes were obtained from the spleens of immunized mice by density gradient centrifugation using Lymphoprep (specific gravity 1.077) (Sigma-Aldrich) as directed by the manufacturer. Single splenocyte suspensions were prepared in complete RPMI 1640 containing 10% fetal bovine serum and 1% penicillin-streptomycin L-glutamine (Gibco, Carlsbad, CA, USA) at a final concentration of 2 × 106 cells/mL.
4
Induction of Experimental Autoimmune Uveitis in Mice
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The method used to induce tEAU has been reported previously [4 (link)]. Briefly, B6 mice were immunized with IRBP1–20 in adjuvant, and 11 days later, T cells were purified from the draining lymph nodes and spleen by passage through a nylon wool column, then 1 × 107 cells in 2 ml of RPMI 1640 medium were added to each well of a six-well plate (Costar, Corning, NY, USA) and stimulated with 20 μg/ml of IRBP1–20 in the presence of 1 × 107 irradiated syngeneic spleen cells as APCs. After 2 days, the activated lymphoblasts were isolated by gradient centrifugation on Lymphoprep (Sigma-Aldrich) and injected i.p. in 0.2 ml of PBS into naive B6 recipients (5 × 106 cells/mouse). Experimental mice were killed on day 15, unless stated otherwise.
5
Murine Splenocyte Isolation and Analysis
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Twenty days after tumor transplantation, the mice were sacrificed and the splenocytes were obtained via passing the spleens through 100 μm nylonmesh filters. Lymphocytes were isolated on a density gradient using Lymphoprep™ (Sigma-Aldrich, UAS). These cells were then washed once with PBS (pH 7.3) and adjusted to a final concentration of 1 × 106 cells/mL, followed by incubation with mouse monoclonal anti-CD4 conjugated phycoerythrin and anti-CD8 conjugated allophycocyamin antibodies on ice for 2 h in the dark. All cells were finally re-suspended in 300 μL of PBS and analyzed by flow cytometry.
6
Chicken Immune Cell Isolation and Stimulation
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PBMCs and splenocytes were prepared from 30-day-old SPF chickens. PBMCs were isolated from peripheral blood using Ficoll-Hypaque (Sigma) density sedimentation. Splenic lymphocytes were obtained from the spleens of birds via density gradient centrifugation by using Lymphoprep (specific gravity 1.077; Sigma) according to the manufacturer’s instructions. PBMCs and splenocytes were suspended and cultured in 24-well microtiter plates filled with complete Roswell Park Memorial Institute 1640 medium containing 10% FBS and 1% penicillin-streptomycin/L-glutamine (Gibco, Carlsbad, CA, USA) at a final concentration of 2 × 106 cells/mL. Cells were treated with 3 μg/mL HA1–2 or 10 μg/ml HA1–2-fliC (containing 3 μg HA1–2 according to the molecular weight). After incubation for 12 h, cells were harvested for RNA extraction. Cytokine and chemokine production by cells was evaluated via qRT-PCR.
7
Isolation and Characterization of PBMCs from Acute Laryngitis Patients
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Peripheral blood (6 ml) was obtained from a patient with acute laryngitis in tubes containing EDTA. The patient signed a written consent to participate in this study (the sample was taken on April 17th 2019). Peripheral blood mononuclear cells (PBMCs) were purified using Lymphoprep (Sigma-Aldrich; Merck KGaA) and centrifuged at 50 × g for 30 min at room temperature. Cells were maintained in DMEM medium (1 ml; cat. no. 11965-118, Gibco; Thermo Fisher Scientific, Inc.) without serum. Cell viability was analyzed using trypan blue and a cell suspension (7.5×105 viable cells/ml) was prepared. The experiment was performed according to the appropriate guidelines for human use approved by the Institutional Committee of Bioethics of the Escuela Nacional de Ciencias Biológicas-IPN.
8
Lymphocyte Radiation Response Protocol
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Lymphocyte extraction and treatment were conducted as previously described.17 (link) In summary, primary lymphocyte cells were obtained from peripheral blood using LymphoPrep (Sigma) and short-term cultured for 6 days in RPMI-1640 with l -glutamine (Biological Industries), supplemented with interleukin-2, 15% fetal calf serum, 1% 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, and 1% penicillin/streptomycin. One day following lymphocyte extraction, half of each sample was γ-irradiated with 8 Gy at a high-dose rate (0.86 Gy/min, Orthovoltage X-ray machine), as previously described.19 (link) Although this dose exceeds the portion used in classical radiotherapy or screening radiation, the DNA damage it causes presumably mimics the additive effect of repeated radiation exposure, which usually occurs in the clinic.9 (link) Additionally, we chose this dose based on our previous study showing differences in gene expression in BRCA mutation carriers after ionizing radiation.17 (link)
9
Isolation and Cryopreservation of PBMCs
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Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood by density gradient centrifugation using Lymphoprep [Stem Cell Technologies, 07851] as per to manufacturer’s instructions. Briefly, 10 mL of whole blood were diluted with 10 mL of Dulbecco’s phosphate-buffered saline (dPBS) (Sigma-Aldrich, Saint Louis, MI, USA, D8537-500ML) and layered over 10 mL of Lymphoprep; then centrifuged at 800× g for 20 min without brake and acceleration. The lymphocyte layer was collected and frozen in 5 million cells aliquots at −80 °C in a solution of 10% dimethyl sulfoxide (DMSO) (Sigma-Aldrich, Saint Louis, Missouri USA, 472301) diluted in fetal calf serum (FCS) (Gibco, Waltham, MA, USA 10082147) until analysis.
10
Isolation of Splenic Lymphocytes
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Two weeks after the third immunization, splenic lymphocytes were obtained from the spleens of mice by density gradient centrifugation using Lymphoprep (specific gravity 1.077) (Sigma-Aldrich) according to the manufacturer’s instructions. Single-splenocyte suspensions were prepared in complete Roswell Park Memorial Institute (RPMI) 1640 medium plus 1% penicillin-streptomycin/l -glutamine and 10% fetal bovine serum (Gibco, Carlsbad, CA, USA) at a final concentration of 2 × 106 cells/ml.
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