Murine BMDM Itgam/CD11b CRISPR-Cas9 optimization screen Day 5: BMDMs were resuspended in nucleofection solutions forprimary cells (Primary Cell Optimization 96-well Nucleofector Kit, Lonza, V4SP-9096) at a density of 5 × 105 cells per reaction in 20 µl nucleofection solution and mixed with Cas9-RNP containing B2M targeting gRNAs. This mixture was nucleofected using the Lonza 4D Nucleofector (4D-Nucleofector Core Unit: Lonza, AAF-1002B; 4D-Nucleofector X Unit: AAF-1002X). Immediately after nucleofection, ~180 µl of prewarmed BMDM culture medium was added to each well, and cells were harvested by gently washing the well. Each reaction was transferred to a single well in a 6-well TC-treated plate containing 2 ml of prewarmed BMDM culture medium.
Murine monocyte culture and eGFP CRISPR-Cas9 optimization screen: BM was harvested from tibias and femurs of eGFP-transgenic mice. Red blood cells were lysed with ACK lysis buffer. Monocytes were isolated using a negative selection kit (Miltenyi). Cells were washed once with 1× PBS and resuspended in the nucleofection solutions for primary cells (Primary Cell Optimization 96-well Nucleofector Kit, Lonza) at a density of 2 × 105 cells per well. Cells were nucleofected as above and immediately transferred to 6-well TC-treated plates containing prewarmed BMDM culture medium.
CRISPR-Cas9-mediated KO in murine BMDCs: BMcells were prepared, and red blood cells were lysed with ACK lysis buffer. BM cells were washed twice with 1× PBS and electroporated in the appropriate primary nucleofection solution (Primary Cell Optimization 4D-Nucleofector X Kit, P3 Primary Cell 4D-Nucleofector X Kit) using the Lonza 4DNucleofector (4DNucleofector Core Unit: Lonza, AAF-1002B; 4D-Nucleofector X Unit: AAF-1002X) as described above. Specifically, 2 × 106 BM cells per reaction were resuspended in 20 µl of primary nucleofection solution and mixed with Cas9-RNP containing targeting or NTC gRNAs.
Optimized conditons are then also transferred to the human monocytes and macropahages /DCs. please see table in reference.