Profection mammalian transfection system calcium phosphate

We generated a Lenti virus expressing CAS9 and a sgRNA targeting CD19. The sgRNA sequence 5′-TGGAATGTTTCGGACCTAGGTGG-3′ (19 (link)) was cloned into pL-CRISPR.EFS.GFP (addgene Plasmid #57818 - Lentiviral CRISPR-Cas9 delivery for SpCas9 and sgRNA. Co-expresses eGFP via P2A cleavage site). Envelope plasmid was pMD2.G (addgene Plasmid #12259) and packaging plasmid psPAX2 (addgene Plasmid #12260). Plasmids were transfected to 293T cell line using the calcium Phosphate Profection Mammalian Transfection System (Promega cat#E1200). Viral supernatant was used for infection of Nalm6 cells with addition of Polybrene (8 ng/ml). CD19 positive cell were depleted using PE conjugated anti-CD19 antibody and anti-PE magnetic beads, on a MACS LD depletion column (Miltenyi), followed by single cell culture by plating. CD19 negative single-clones were confirmed by flow cytometry and wells were sequenced for the CD19 KO locus.

Approximately 10 000 293T cells were seeded per well of a white-walled 96-well plate (Thermo-Scientific Nunc), previously coated in Geltrex LDEV-free reduced growth factor basement membrane matrix (Gibco) according to the manufacturer's thin layer non-gelling method to enhance cell adhesion. The next day, the cells were transfected using the calcium-phosphate profection mammalian transfection system (Promega). Transfection solution was prepared with the appropriate ACE2 plasmid following manufacturer instructions for 10 µg plasmid per 10 cm dish. To proportionally reduce volume added according to relative surface area, 4 µl of solution were added per well of the 96-well plate. Then, 24 h after transfection, the cells were infected with pseudovirus following a previously published protocol [30 (link)]. The spent medium was removed, and 100 µl of fresh media containing the indicated dilution of pseudovirus was added per well. Plates were spinoculated for one hour at 330 g. At 18–24 h post infection, media was removed, cells were washed with PBS, and cells were lysed using Renilla luciferase assay system lysis buffer (Promega) and one freeze–thaw cycle. The lysate was assayed for luminescence using the Renilla luciferase assay system (Promega) on a Synergy H1 microplate reader (BioTek).

A previously published protocol [30 (link)] was followed. First, 3–5 × 10⁶ 293T cells were seeded into 10 cm dishes. The next day, cells were transfected with the appropriate spike plasmid (Wuhan-Hu-1: BEI NR-52310; Delta: Addgene no. 185593, a gift from Marceline Côté, [31 (link)]; Omicron: Addgene no. 185452, a gift from Marceline Côté, [32 (link)]) using the calcium-phosphate profection mammalian transfection system (Promega). All spike plasmids were human-codon optimized and coded for the full-length protein. There were 20 µg of plasmid used per dish. Approximately 20 h later, media was removed and cells were infected with 50 µl vesicular stomatitis virus strain VSVΔG-Rluc decorated with VSV-G (seed particles obtained from Benhur Lee, Icahn School of Medicine at Mt Sinai; stocks renewed using pCMV-VSV-G, Addgene no. 8454, a gift from Bob Weinberg, [33 (link)]) in 5 ml media. After 1 h incubation, cells were washed with phosphate-buffered saline (PBS) and 8 ml fresh media containing 1.6 µg of antibody neutralizing against VSV-G (Millipore Sigma no. MABF2337-25UG) were added. The supernatant was harvested 24 h post infection and aliquots were stored at −80°C.