Miraclean endotoxin removal kit

Heavy- and light-chain genes were ligated into the Mammalian PowerExpress System (Toyobo Co., Ltd.), and this construct was used as the antibody expression vector^{28 (link)}. Endotoxins of the plasmid were removed using MiraCLEAN Endotoxin Removal Kit (Mirus Bio LLC, Madison, WI, USA) twice after it was linearised with SspI, and then the plasmids were transfected into CHL-YN and CHO-K1 cells using PEI as described above. IgG-expressing cells were selected and cultured in medium containing 5 μg/mL puromycin (InvivoGen, San Diego, CA, USA).

The CRISPR Cas9 system was used to knockout AIF1 in Lin⁻CD117⁺ HSC or total BM. Two CRISPR DNA plasmids were created using the GeneArt CRISPR Nuclease Vector Kit (Thermo Fisher) to target AIF1. The gRNA sequences were: 5′-AGAGTAGCTGAACGTCTCCT-3′ (pAIF1-T3) and 5′-GCTGAAGAGATTAATTAGAG-3′ (pAIF1-T4). Control plasmids contained scrambled targeting sequences (pControl). Plasmids were purified using PureLink™ HiPure Plasmid Maxiprep Kit (Thermo Fisher) and cleaned with the MiraCLEAN® Endotoxin Removal Kit (Mirus, Madison WI). Twenty microgram of control or AIF1 targeting plasmids were electroporated using a square wave electroporator under the following conditions: 230 V, 4 ms, 5 pulses.

The CRISPR Cas9 system was used to knockout AIF1 in total BM or CD11b⁺Ly6C/G⁺CD115⁺MHC class II^neg. The CRISPR DNA plasmid was created using the GeneArt CRISPR Nuclease Vector Kit (Thermo Fisher) to target AIF1, as previously described by Elizondo et al^{12 (link)}. The gRNA sequences are: 5′-GCTGAAGAGATTAATTAGAG-3′ (pAIF1). Control plasmids contained scrambled targeting sequences (pControl). Plasmids were purified using PureLink HiPure Plasmid Maxiprep Kit (Thermo Fisher) and cleaned with the MiraCLEAN Endotoxin Removal Kit (Mirus, Madison WI). Cells were transfected with 40 μg of control or AIF1 targeting plasmids using a square wave electroporator with the following parameters: 230 V, 4 ms, 5 pulses. For small interfering RNA (siRNA)-mediated silencing of cells, 0.5 nmol of the oligonucleotide sequence 5′-GGCAAGAGAUCUGCCAUCUUG-3′ was electroporated under the following settings: 310 V, 10 ms, 1 pulse.