A31604 02

Adipose tissue samples were washed several times with phosphate-buffered saline (PBS, Basalmedia, B310KJ) containing penicillin (1000 U/mL, Basalmedia, S110JV) and streptomycin (1000 μg/mL, Basalmedia, S110JV) and then digested with 0.075% type I collagenase (Sigma, C0130) for 40 min. The digested adipose tissue was filtered through a 100-mesh filter and the resulting suspension was centrifuged to obtain cell precipitates. The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, C11885500BT) containing 10% fetal bovine serum (FBS, Gibco, A31604-02), penicillin (100 U/mL), and streptomycin (100 μg/mL). The cells were subcultured (passaged) when the cellular confluence reached approximately 80% and adMSCs from the third passage (P3) were used in subsequent experiments.
adMSCs identification was performed as previously described by Wang et al. [11 (link)]. Briefly, the immunophenotype of P3 adMSCs was determined by evaluating cluster of differentiation 34 (CD34), CD44, CD45, CD105, CD106, and CD166 using flow cytometry, and osteogenic and adipogenic differentiation was performed to confirm their multipotency.

L-929 cells (CVCL_0462) were cultured in DMEM (12430-054; Gibco), 10% horse serum (HS; 26050-070; Gibco), 0.5 mM L-glutamine (25030-081; Gibco), and 1% penicillin/streptomycin (P/S; 15140-122; Gibco).
For mixed-lineage kinase domain-like (Mlkl) silencing, L929 cells were infected with lentivirus-containing shRNA against Mlkl (360819; Sigma Mission) at a multiplicity of infection (MOI) 100. Four days later, cells were harvested and protein extraction was performed to assess knock-down efficiency.
For inducing MLKL phosphorylation (p-MLKL), L929 cells were treated with 30-ng/ml human recombinant TNF-α (cyt-223-b; Prospec), 100 nM smac mimetic (TL-32711; A1901; Active Biochem), and 20 μM zVAD-fmk (V116; Sigma; TSZ) for 1 h, and then protein was extracted.
NIH 3T3 cells (CVCL_0594), were cultured in DMEM (12430-054; Gibco), 10% fetal bovine serum (FBS; A31604-02; Gibco), 0.5 mM L-glutamine (25030-081; Gibco), and 1% P/S (15140-122; Gibco). This cell line expresses high levels of MLKL and was used as positive control for testing anti-MLKL antibody.

Acute Lymphoblastic Leukemia cell line (Nalm-6, NCBI C212) was obtained from the Pasteur Institute collection, Tehran, Iran. Nalm-6 cells were cultured in RPMI-1640 with 2 mM l-glutamine (Gibco™ A1049101) containing 10% fetal bovine serum (FBS) (Gibco™ A3160402), 1% antibiotic (Penicillin–Streptomycin Solution 100X, Biowest, L0022) in a humidified atmosphere of 5% CO₂ incubator at 37 °C. ZME was prepared according to methanolic extract protocol that Saedi Dezaki et al. applicated in their study^{21 (link)}. ZME lyophilized powder was dissolved in dimethylsulfoxide (DMSO) (Merck, CAS 67-68-5) and culture media as main ZME stock, and diverse concentrations of ZME were diluted and obtained from main solution stock. The stock of doxorubicin (EBEWE Pharma, Austria) was also diluted into considered concentration for treatment.