T-cell ELISPOTS were performed as previously described [44 (link)]. Briefly, splenocytes from AAV9-SaCas9-injected mice were isolated and purified using Lympholyte M (Cederlane). 250,000 cells were mixed with either Cas9 protein (Applied Biological Materials) or cell media only as a negative control, and subsequently plated in a 96-well ELISPOT plate (Millipore, MSIPS4510) in 100 µL per well. Stimulation was performed with 0.02 µg/µL of Cas9 protein at 37ºC in a humidified incubator, 7% (v/v) CO2, for 40 h. Mouse IFN-γ ELISPOT pairs (3321–3-250 and 3321–6-250) and Streptavidin-alkaline phosphatase (3310–10) were purchased from Mabtech. Spots were developed using substrate Sigmafast BCIP/NBT (Sigma, B5655). Plates were shipped to Zellnet Consulting and enumerated using a Zeiss KS ELISPOT system. Full plates images are reported in Extended Data Fig. 4 .
Cas9 protein
Manufactured by Mabtech
The Cas9 protein is a key component of the CRISPR gene-editing system. It functions as a DNA-binding and DNA-cleaving enzyme that can be programmed to target specific DNA sequences for modification. The Cas9 protein is derived from the bacterial CRISPR-Cas system and plays a central role in the gene-editing process.
Automatically generated - may contain errors
Lab products found in correlation
Streptavidin alkaline phosphatase, by Mabtech (2 mentions)
Msips4510, by Merck Group (2 mentions)
Mouse ifn γ elispot pairs 3321 3 250, by Mabtech (2 mentions)
Ks elispot system, by Zeiss (2 mentions)
Sigmafast bcip nbt, by Merck Group (2 mentions)
Cas9 protein, by Applied Biological Materials (2 mentions)
2 protocols using cas9 protein
1
Quantifying T-cell Responses to Cas9 Protein
2
Quantifying T-cell Responses to Cas9 Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
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T-cell ELISPOTS were performed as previously described [44 (link)]. Briefly, splenocytes from AAV9-SaCas9-injected mice were isolated and purified using Lympholyte M (Cederlane). 250,000 cells were mixed with either Cas9 protein (Applied Biological Materials) or cell media only as a negative control, and subsequently plated in a 96-well ELISPOT plate (Millipore, MSIPS4510) in 100 µL per well. Stimulation was performed with 0.02 µg/µL of Cas9 protein at 37ºC in a humidified incubator, 7% (v/v) CO2, for 40 h. Mouse IFN-γ ELISPOT pairs (3321–3-250 and 3321–6-250) and Streptavidin-alkaline phosphatase (3310–10) were purchased from Mabtech. Spots were developed using substrate Sigmafast BCIP/NBT (Sigma, B5655). Plates were shipped to Zellnet Consulting and enumerated using a Zeiss KS ELISPOT system. Full plates images are reported in Extended Data Fig. 4 .
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