4d nucleofector ds 137

MM.1 S cells, kindly provided by Leif Bergsagel, were modified to MM.1S-CG [16 (link)]. ΔCS1-MM.1S-CGs were generated using the CS1 guide sequence above cloned into pMLM3636 (Addgene) lentiviral vector. MM1.S-CG cells were transduced with 1ug gRNA plasmid, 250 ng Cas9-HF plasmid and electroporated in SF solution using the Lonza 4D Nucleofector DS-137 program. CS1 negative cells were sorted using a MoFlow. OPM2 cells (DMSZ, Germany) were modified to express CBR-GFP as above.

The Itga4 KO MM1.S-GFP were generated by using CRISPR-Cas9. Briefly, the α₄ knockout guide RNA sequence was cloned into the MLM3636 plasmid (Addgene). 250,000 MM.1S cells were resuspended in 20μL SF solution (Lonza), 1 μg gRNA plasmids and 250 ng Cas9-HF plasmid (Addgene # 43,945 pc3 CAS9hc) and electroporated using the Lonza 4-D Nucleofector DS-137 program (optimized for these cells, not shown). α₄ negative cells were sorted using a MoFlow sorter.
Six to ten-week old NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) were used in all MM.1S mice experiments. Briefly, 500,000 MM.1S parent or alpha4 KO cells were injected i.v. into tail veins of mice. Tumors were allowed to grow for ~ 2–3 weeks and tumor progression was followed by bioluminescence imaging. Flow cytometry for CD138 (Biolegend) was used to assess percent MM.1S cells in multiple tissues. Flow was run on an Attune and analyzed using FlowJo (Treestar).