Red blood cell lysis buffer

The femurs and tibias from mice were collected, and bone marrow cells were flushed with Hank's (Hyclone) contained with 5% penicillin streptomycin combination, then cell suspensions were filtered with 100μm cell sterile strainer (Falcon) for removing cell clumps. Cell precipitates were collected by centrifugation and suspended in Hank's. Cell suspensions treated with red blood cell lysis buffer (TIANGEN) for a few minutes and PBS (Hyclone) washed once. Finally, cell suspensions were suspended with RPMI 1640 Medium (Hyclone) contained with 20% fetal bovine serum, 1% penicillin streptomycin combination, 1% L-glutamine and 50ng/mL MCSF (315-02, PeproTech), plated onto 10 cm cell culture dishes. About after three days, cells were digested using trypsin, centrifuged to obtain cell precipitates. Cell precipitates were suspended with RPMI 1640 Medium contained with 10% fetal bovine serum, 1% penicillin streptomycin combination, 1% L-glutamine and 50ng/mL MCSF 19 (link). After about two days BMDMs could be obtained. BMDMs were treated with 100ng/mL LPS (L2880, Sigma) + 20ng/mL IFN-γ (315-05, PeproTech) or 10ng/mL IL-4 (214-14, PeproTech) + 10ng/mL IL-13 (210-13, PeproTech), and collected mRNAs for qRT-PCR and RNA-Seq analysis. BMDMs were treated with 100ng/mL LPS or 10ng/mL IL-4 + 10ng/mL IL-13, and collected proteins for WB analysis. 20 (link), 21 (link)

Two milliliters of venous blood were collected into an ethylene diamine tetraacetic acid anticoagulant tube, transported immediately to the Children’s Hospital within 2 h, then centrifuged at 3000 rpm for 3–5 min to separate the plasma and blood cells. A volume of 200 μL of plasma was stored at −80 °C to measure the retinol concentrations within 1 week. After abundant lysis with red blood cell lysis buffer (TIANGEN, Beijing, China), white cell were separated from whole blood cells. Then the RNA in the white cells was protected in 1 mL of Trizol Reagant (Ambion, Carlsbad, California, USA) at −80 °C for future use.

Human peripheral blood samples of 164 individuals from longevous families were collected from Hainan Province in southern China in two batches, 50 samples [5 (link)] and 114 samples, respectively. The 164 biospecimens included 71 long-lived individuals (LLI, age: 98.3 ± 3.4 year), 57 offspring of LLIs (F1, age: 60.9 ± 6.8 year) and 36 spouses of F1 (F1SP, age: 59.3 ± 5.8 year). White blood cells were isolated from the peripheral blood using red blood cell lysis buffer (Tiangen, Beijing, China) and centrifugation at 4000 rpm for 10 min at room temperature. Total RNA samples were extracted using the TRIzol method. The rRNA-depleted RNA-seq libraries were prepared following the instructions contained in the Ribo-Zero kit (China) and were deeply sequenced using the Illumina HiSeq 4000 platform. All research protocols were approved by the Ethics Committee at the Kunming Institute of Zoology, Chinese Academy of Sciences (Approval Code: SMKX-20141220-74; Approval Date: 20 December 2014).