Rpmi 1640 medium

On days 0~5, hematopoietic stem and progenitor cells (HSPCs) were massively amplified in StemPro™−34 SFM medium (10639011; life technologies) supplemented with 1% penicillin–streptomycin, 10% FBS (SE100-B; VISTECH), 1% GlutaMAX (35050061; Gibco), 50 ng/mL G-CSF (10007-HNCH-5; Sino Biological), and 20 ng/mL hIL3. On days 5~9, the cells were cultured in RPMI 1640 medium (C11875500BT; ThermoFisher) supplemented with 1% penicillin–streptomycin, 10% FBS, 1% GlutaMAX, and 100 ng/mL G-CSF. The medium was changed every 2~–3 days. Cells differentiated up to day 9 were collected for subsequent experimental assays.

To this end, 14 days after the last vaccination, three mice in each experimental group were euthanized and lymphocytes were harvested under aseptic conditions for cytokine evaluation [47 ]. The harvested lymphocytes were cultivated in RPMI-1640 medium (Gibco) supplemented with 20% fetal bovine serum (FBS). Next, 500 μL of extracted lymphocyte suspension of each mouse was cultured in two wells of a 24-well cell culture plate, challenged with 50 μg of crude antigen prepared [48 (link)], and the volume of each well was reached to 1 mL using complete RPMI medium. The plates were incubated for 48–72 h at 37°C with 5% CO₂. Finally, the supernatant of each well was harvested and centrifuged (3000 rpm, 10 min); then, interferon γ (IFN-γ) and interleukin 4 (IL-4) cytokines were measured in triplicate by commercial ELISA kits (Karmania Pars Gene, Kerman, Iran), based on the manufacturer's protocol.

After the mice were euthanized and soaked with 75% ethanol for 1 min, splenic tissues were harvested into the 10‐cm cell‐culture dish. Four to five milliliters of mice lymphocyte isolate (cat#: 7211011, Dakewe) was taken, and the splenic tissues were grinded using the 2‐mL syringe plunger, followed by filtration with the 40‐µm filter. Thereafter, we added the spleen cell suspension into the 15‐mL centrifuge tube at once, introduced 1 mL RPMI‐1640 medium (cat#: 42401042, Gibco), and centrifuged the mixture at 800 × g at 4°C for a 30‐min duration. Afterward, the solution within centrifuge tube was separated in 4 layers, including the RPMI‐1640 medium, lymphocyte, separation medium layers, and the layer containing red blood cells as well as cell debris, from the top down. After aspiration, we added the lymphocyte layer to 10 mL RPMI‐1640 medium, centrifuged the mixture for a 10‐min period at 250 × g and 4°C to harvest lymphocytes, and suspended cells within serum‐free medium. Mouse IFN‐γ (cat#: 3511–4hpw‐2, Mabtech) and IL‐2 ELISpot plus kits (cat#: 3441–4hpw‐2, Mabtech) were then adopted for detecting interferon gamma (IFN‐γ)‐ and interleukin (IL)‐2‐positive cells. 96‐Well PVDF plates were rinsed by PBS (200 µL) twice, prior to 2‐h blocking using RPMI‐1640 medium that contained 10% fetal bovine serum under 25°C. We later added the separated lymphocytes (2.5 × 10⁵ cells) into each well and treated them with 1 µg/mL of S protein peptide pools from the EG.5 (GenScript, Nanjing, China), XBB.1.5 (cat#: DD9123, Vazyme), and BA.4/5 (cat#: DD 9125, Vazyme) subvariants for 24 h under 37°C. Lymphocytes were later subjected to 2‐h incubation using anti‐Mouse IFN‐γ or anti‐mouse IL‐2 antibody under ambient temperature and then 1‐h incubation using streptavidin‐horseradish peroxidase (1:1000). Cells were later washed before 5‐min incubation using 3,5,‐tetramethylbenzidine (TMB, 100 µL) substrate solution until visible spots appeared. Employ the ImmunoSpot S6 Universal spot counter for imaging and counting of IFN‐γ‐secreting cell spots.

Because of the clonal instability of the PC12 cell line (Fujita et al. 1989 (link)), the experiments were performed on cells that had undergone fewer than five passages, and all studies were repeated several times with different batches of cells. As described previously (Crumpton et al. 2000a (link); Qiao et al. 2003 (link); Song et al. 1998 (link)), PC12 cells (1721-CRL; American Type Culture Collection, Manassas, VA) were seeded onto 100-mm poly-d-lysine-coated plates in RPMI-1640 medium (Invitrogen, Carlsbad, CA) supplemented with 10% inactivated horse serum (Sigma Chemical Co., St. Louis, MO), 5% fetal bovine serum (Sigma Chemical Co.), and 50 μg/mL penicillin streptomycin (Invitrogen). Cells were incubated with 7.5% CO₂ at 37°C, and the medium was changed every 2 days. For studies in the undifferentiated state, cells were seeded at varying densities so that, regardless of the total time of incubation, the cells would reach a final confluence of 60–70%. Twenty-four hours after seeding, the medium was changed to include the various test substances: chlorpyrifos (Chem Service, West Chester, PA), diazinon (Chem Service), parathion (Chem Service), physostigmine (Sigma Chemical Co.), dieldrin (Chem Service), or NiCl₂ (Sigma Chemical Co.). Because of their poor water solubility, the pesticides were dissolved in dimethyl sulfoxide (Sigma Chemical Co.), achieving a final concentration of 0.1% in the culture medium; accordingly, all cultures included this vehicle, which had no effect on the PC12 cells (Qiao et al. 2001 (link), 2003 (link); Song et al. 1998 (link)).
For studies in differentiating cells, 3 × 106 cells were seeded; 24 hr later, the medium was changed to include 50 ng/mL 2.5 S murine NGF (Invitrogen), and each culture was examined under a microscope to verify the subsequent outgrowth of neurites. The test agents were added concurrently with the start of NGF treatment.

As described previously (Crumpton et al. 2000 (link); Qiao et al. 2003 (link); Song et al. 1998 ), PC12 cells (1721-CRL; American Type Culture Collection, Rockville, MD) were grown in RPMI-1640 medium (Invitrogen, Carlsbad, CA) supplemented with 10% inactivated horse serum (Sigma Chemical Co., St. Louis, MO), 5% fetal bovine serum (Sigma), and 50 μg/mL penicillin–streptomycin (Invitrogen); cells were incubated with 7.5% CO₂ at 37°C, and the medium was changed every 2 days. Because of the clonal instability of the PC12 cell line (Fujita et al. 1989 (link)), the experiments were performed on cells that had undergone fewer than five passages, and studies were repeated several times with different batches of cells. For studies in the undifferentiated state, 3–6 × 10⁶ cells were seeded onto 100-mm poly-l-lysine–coated plates, and 24 hr later the medium was changed to include 5 or 50 μM CPF (purity, 98%; Chem Service, West Chester, PA). CPF was dissolved in dimethyl sulfoxide (DMSO), achieving final DMSO concentrations of 0.1–1% in the culture medium, and the corresponding control samples contained equivalent DMSO concentrations. Preliminary studies were conducted to verify that these concentrations of DMSO had no effect on the measured parameters in PC12 cells (data not shown), in agreement with earlier work (Qiao et al. 2001 (link), 2003 (link); Song et al. 1998 ).
For studies during differentiation, 3.5 × 10⁶ cells were seeded; 24 hr later the medium was changed to include 50 ng/mL NGF (Sigma), and over the ensuing week, each culture was examined under a microscope to verify the outgrowth of neurites. CPF or physostigmine (Sigma) was added either simultaneously with the addition of NGF or after a 4-day NGF pretreatment.