Collagenase type cls 4

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Collagenase type CLS IV is an enzyme used for the dissociation of connective tissues. It is effective in the isolation of a variety of cell types, including those derived from epithelial, endothelial, and neural tissues.

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Lab products found in correlation

Cd3 pe, by Miltenyi Biotec (2 mentions) Hanks balanced salt solution ca2 mg2, by Thermo Fisher Scientific (2 mentions) Cd14 percp, by Miltenyi Biotec (2 mentions) Ccr7 pe vio770, by Miltenyi Biotec (2 mentions) Cd4 vioblue, by Miltenyi Biotec (2 mentions) Cd8 percp, by Miltenyi Biotec (2 mentions) Cd20 percp, by Miltenyi Biotec (2 mentions) Cd45ro apc, by Miltenyi Biotec (2 mentions) Anti cd3 okt3, by Miltenyi Biotec (1 mentions)

Spelling variants of this product

Collagenase (6 citations) Collagenase CLS (2 citations) Collagenase IV (2 citations)

2 protocols using collagenase type cls 4

Single-cell expansion and antigen-specific T-cell analysis

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Up to 4 biopsies per patient were pooled and digested in 1 mL of Hanks’ balanced salt solution Ca²⁺Mg²⁺ (Gibco) supplemented with 0.5% (v/v) human AB-serum, 100 IU ml⁻¹ of penicillin, 100 µg ml⁻¹ of streptomycin, 0.25 µg ml⁻¹ of amphotericin B, 10 IU ml⁻¹ of DNase I (CellSystems) and 1 mg ml⁻¹ of collagenase type CLS IV (Biochrom) for 30 min at 37 °C while shaking. Cells were filtered through a 40-µm cell strainer to remove aggregates and stained with fluorochrome-conjugated antibodies to CD4-VioBlue, CD3-PE, CD8-PerCP, CD14-PerCP, CD20-PerCP, CD45RO-APC and CCR7-PE-Vio770 (all Miltenyi Biotec). Each 200 CD3⁺CD4⁺CD45RO⁺ cells were sorted in multiple wells of a 96-well plate containing irradiated allogeneic feeder cells in TexMACS medium, supplemented with 5% (v/v) human AB-serum, 200 U ml⁻¹ of IL-2 and 100 IU ml⁻¹ of penicillin, 100 µg ml⁻¹ of streptomycin, 0.25 µg ml⁻¹ of amphotericin B at a density of 2 × 10⁵ cells cm⁻². Cells were polyclonally expanded for 4 weeks in the presence of 30 ng ml⁻¹ of anti-CD3 (OKT-3; Miltenyi Biotec). Restimulation with different fungal lysates was performed as described above. To calculate the frequencies of reactive T cells, the mean values were calculated for each antigen and divided by the number of input wells.

Martini G.R., Tikhonova E., Rosati E., DeCelie M.B., Sievers L.K., Tran F., Lessing M., Bergfeld A., Hinz S., Nikolaus S., Kümpers J., Matysiak A., Hofmann P., Saggau C., Schneiders S., Kamps A.K., Jacobs G., Lieb W., Maul J., Siegmund B., Seegers B., Hinrichsen H., Oberg H.H., Wesch D., Bereswill S., Heimesaat M.M., Rupp J., Kniemeyer O., Brakhage A.A., Brunke S., Hube B., Aden K., Franke A., Iliev I.D., Scheffold A., Schreiber S, & Bacher P. (2023). Selection of cross-reactive T cells by commensal and food-derived yeasts drives cytotoxic TH1 cell responses in Crohn’s disease. Nature Medicine, 29(10), 2602-2614.

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Expansion and Characterization of Antigen-Specific T Cells

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Up to 4 biopsies per patient were pooled and digested in 1 mL of Hanks’ balanced salt solution Ca²⁺Mg²⁺ (Gibco) supplemented with 0.5% (v/v) human AB-serum, 100 IU ml⁻¹ of penicillin, 100 μg ml⁻¹ of streptomycin, 0.25 μg ml⁻¹ of amphotericin B, 10 IU ml⁻¹ of DNase I (CellSystems) and 1 mg ml⁻¹ of collagenase type CLS IV (Biochrom) for 30 min at 37 °C while shaking. Cells were filtered through a 40-μm cell strainer to remove aggregates and stained with fluorochrome-conjugated antibodies to CD4-VioBlue, CD3-PE, CD8-PerCP, CD14-PerCP, CD20-PerCP, CD45RO-APC and CCR7-PE-Vio770 (all Miltenyi Biotec). Each 200 CD3⁺CD4⁺CD45RO⁺ cells were sorted in multiple wells of a 96-well plate containing irradiated allogeneic feeder cells in TexMACS medium, supplemented with 5% (v/v) human AB-serum, 200 U ml⁻¹ of IL-2 and 100 IU ml⁻¹ of penicillin, 100 μg ml⁻¹ of streptomycin, 0.25 μg ml⁻¹ of amphotericin B at a density of 2 × 10⁵ cells cm⁻². Cells were polyclonally expanded for 4 weeks in the presence of 30 ng ml⁻¹ of anti-CD3 (OKT-3; Miltenyi Biotec). Restimulation with different fungal lysates was performed as described above. To calculate the frequencies of reactive T cells, the mean values were calculated for each antigen and divided by the number of input wells.

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