KP1 Cd47 KO and control cells were previously described20 (link). The single guide RNAs (sgRNAs) for CD47, Csf1 and Ccl2 were purchased from Synthego. We added 12 µl of SE buffer (Lonza, V4XC-1032) to each well of a 96-well v-bottom plate. Then 3 µl of sgRNA (300 pmol) was added to the SE buffer. An aliquot of 0.5 µl of Alt-R S.p. Cas9 (Integrated DNA Technologies, 1081059) was added to 10 µl of SE buffer. Next, Cas9 was added to the sgRNA solution, mixed thoroughly and incubated at 37 °C for 15 min to form the ribonucleoproteins. NJH29 cells or KP1 cells were pelleted, counted and resuspended to 106 cells per reaction in 5 µl of SE buffer. Cells and the ribonucleoprotein solution were added to each nucleofection well chamber. Cells were immediately nucleofected using the Lonza 4D-NucleofectorTM X Unit (Lonza, AAF-1002X) with the EN150 program. After nucleofection, warm RPMI medium was added to the cells. The cells were incubated at 37 °C for 15 min and then transferred to a 24-well plate with 1 ml RPMI medium. Editing efficiency was evaluated 4 d later by FACS or RT–qPCR.
V4xc 1032
Manufactured by Lonza
The V4XC-1032 is a versatile laboratory equipment designed for a wide range of applications. It features advanced functionality and precise control capabilities to support various experimental and analytical tasks. The core function of this product is to provide a reliable and adaptable platform for scientific investigations and testing procedures.
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Lab products found in correlation
7 protocols using v4xc 1032
1
CRISPR-Cas9 Gene Editing Protocol
2
Efficient CRISPR-Cas9 Genome Editing
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Three sgRNAs were designed to hybridize approximately 150 bases apart on the target of interest (see Key Resources) and were synthesized by Synthego. The three sgRNAs were resuspended in Tris-EDTA and were mixed at a 1:1:1 ratio. First, 12 μL of SE buffer (Lonza, #V4XC-1032) was added to each well of a 96-well v-bottom plate. Then 3 μL of sgRNA (300 pmol for all three sgRNAs) was added to the SE buffer. An aliquot of 0.5 μL of Alt-R S.p. Cas9 (Integrated DNA Technologies, # 1,081,059) was added to 10 μL of SE buffer. Next the Cas9 was added to the sgRNA solution, mixed thoroughly, and incubated at 37°C for 15 min to form the ribonucleoproteins (RNPs). NCI-H82 and NJH29 cells were pelleted, counted, and resuspended to 106 cells per reaction in 5 μL of SE Buffer. Cells and the RNP solution were added to each well. Cells were immediately nucleofected using the Lonza 4D-Nucleofector X Unit (Lonza, #AAF-1002X) with the EN150 program. After nucleofection, warm RPMI media was added to the cells. Cells were incubated at 37°C for 15 min and then transferred to a 24-well plate. Editing efficiency was evaluated 4 days later by FACS or sequencing.
3
Efficient Genome Editing in NCI-H82 Cells
4
Engineered Nalm-6 Cell Lines with CD19/CD22 Modulation
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Nalm-6 cell lines expressing various amount of CD19 and CD22 were generated as described previously [2 ]. Briefly, CD19, CD22, or both were knocked out of Nalm-6 cells using CRISPR Cas9, and negative cells were transduced with a lentiviral vector encoding either a truncated CD19 protein (extracellular and transmembrane domains only, Uniprot P15391). Briefly, 1×106 cells were resuspended in SF buffer (Lonza, V4XC-1032) mixed with 20nM of ribonucleoprotein protein complex formed with CRISPR Cas9 enzyme (Horizon Discovery, CAS12207) and sgRNAs of interest (Horizon Discovery). NALM-6 cells negative for the antigen of interest were sorted by flow cytometry, transduced with the lentiviral vector, subjected to single cell flow sorting to obtain different levels of CD19 and CD22 expression, and expanded for anlaysis. Nalm-6 cells expressing BCMA and/or SLAMF7 were generated by transduction with a lentiviral plasmid encoding for the extracellular and transmembrane domains of BCMA (Uniprot: Q02223) or SLAMF7 (Uniprot: Q9NQ25) and sorted by flow cytometry for purity.
5
Efficient Genome Editing in NCI-H82 Cells
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Control and targeting sgRNAs were generated as previously described.32 (link) Briefly, for each region of interest, three sgRNAs were designed to hybridize approximately 150 bases apart, and 100 pmol of each sgRNA was resuspended in Tris-EDTA (Synthego) and mixed at a 1:1:1 ratio. In a 96-well v-bottom plate, 3 μL of the sgRNA (total 300 pmol) was added to 12 μL of SE buffer (Lonza V4XC-1032). In another well, 0.5 μL of Alt-R® S.p. Cas9 (Integrated DNA Technologies 1081059) was added to 10 μL of SE buffer, and the Cas9 mix was added to the sgRNA solution, mixed thoroughly, and incubated at 37°C for 15 minutes to form the RNP solution. 1 × 106 NCI-H82 cells were resuspended in 5 μL of SE Buffer, and cells were nucleofected with Lonza 4D-Nucleofector™ X Unit (Lonza AAF-1002X) with the EN150 program immediately following addition of the RNP solution. Following the nucleofection, warm RPMI media was added to the cells, and cells were incubated at 37°C for 15 minutes then transferred to a 24-well plate.
6
CRISPR-Cas9 Knockout Validation Protocol
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Genes were knocked out using one to two synthetic sgRNAs (Synthego) in complex with spCas9 (Aldeveron 9214) that were electroporated into cells using a nucleofector system (Lonza V4XC-1032) according to published methods (Hoellerbauer et al. 2020a (link),b (link)). Briefly, 120,000 cells were mixed with either targeting or nontargeting sgRNA:Cas9 complexes in complete SE nucleofector solution. Cell solutions were added to 16-well minicuvette cells and electroporated using program CN-114. Cells were split into two wells, one with doxycycline and one without, and cell numbers were assayed 5–10 d later. KNTC1 knockout by RNP electroporation was validated by analyzing gDNA 7 d after electroporation. gDNA was extracted, and the target locus was amplified by PCR, Sanger-sequenced, and deconvolved using ICE (Synthego).
7
Genetic Manipulation and Proliferation Assay
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Mad1 and Sgo1 FRT/TO/Hygro vectors were a gift from Jennifer DeLuca. The coding DNA sequence for Mad1 and Sgo1 was amplified from cDNA libraries and thus for proliferation retests synthetic sgRNA targeting these genes span intron-exon boundaries to ensure the ectopic copy was not targeted. All other coding sequences were generated as codon optimized and thus sgRNA resistant gBlocks (IDT) and inserted into restriction enzyme linearized pcDNA5 FRT/TO/Hygro by Gibson assembly and sequence verified. sgRNA:Cas9 mediated gene knockout Genes were knocked out using 1-2 synthetic sgRNAs (Synthego) in complex with spCas9 (Aldeveron 9214) that were electroporated into cells using a nucleofector system (Lonza V4XC-1032) according to published methods (Hoellerbauer et al., 2020a; Hoellerbauer et al., 2020b) .
Briefly, 120k cells were mixed with either targeting or non-targeting sgRNA:Cas9 complexes in complete SE nucleofector solution. Cell solutions were added to 16-well mini-cuvettes cells and electroporated using program CN-114. Cells were split into two well, one with doxycycline and one without, then cell numbers were assayed 5-7 days later.
Briefly, 120k cells were mixed with either targeting or non-targeting sgRNA:Cas9 complexes in complete SE nucleofector solution. Cell solutions were added to 16-well mini-cuvettes cells and electroporated using program CN-114. Cells were split into two well, one with doxycycline and one without, then cell numbers were assayed 5-7 days later.
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