AccutaseLz Reconstitution Instructions - 500 ml
AccutaseLZ is a direct replacement for a frozen bottle of Accutase, cell detachment solution. An AccutaseLZ lyophilized vial contains all ingredients found in a frozen bottle of Accutase except for water. The vials are extremely stable at room temperature but should be stored in the refrigerator or freezer. Once reconstituted and sterile filtered, the AccutaseLZ can be stored in the refrigerator or frozen for later use. A reconstituted bottle of AccutaseLZ is stable in the refrigerator for at least one month. The frozen bottle is good 1 year in the freezer.
Once reconstituted, AccutaseLZ works just like Accutase. It is a cell detachment solution of proteolytic and collagenolytic enzymes. It is useful for the routine detachment of all cells including stem cells from standard tissue culture plastic-ware, adhesion coated plastic-ware, and polymers. Accutase performs exceptionally well in detaching cells for the analysis of cell surface markers, virus growth assay, quiescence assays by serum starvation, transformation assays by oncogene transfection, neural crest cell migration assays, cell proliferation, cell haptotaxis, tumor cell migration assays, routine cell passage, production scale-up (bioreactor), and flow cytometry.
Cell lines tested for Accutase application includes fibroblasts, keratinocytes, vascular endothelial cells, hepatocytes, vascular smooth muscle cells, hepatocyte progenitors, primary chick embryo neuronal cells, bone marrow stem cells, adherent CHO and BHK cells, macrophages, 293 cells, L929 cells, immortalized mouse testicular germ cells, MRC5, 3T3, Vero, COS, HeLa, NT2, MG63, M24 and A375 metastatic melanoma, gliomas U251, D54, HT1080 fibrosarcoma cells, Sf9 insect cells, human embryonic stem cells, human mesenchymal stem cells and human neural stem cells. Accutase does not contain mammalian or bacterial derived products.
Intended Use
Accutase is a direct replacement for trypsin cell detachment solution.
For research use only. CAUTION: Not intended for human or animal diagnostic or therapeutic uses. Contact us for cGMP version.
Reconstitution
Precautions
Do not store Accutase at room temperature. It is recommended to thaw Accutase at 4°C overnight or in a bath of cool water. Do not thaw at 37 °C.
Storage & Shelf Life
Store at -20C lyophilized, 2-8C defrosted. 12 month shelf life frozen.
Formulation:1X ACCUTASE enzymes in Dulbecco’s PBS (0.2 g/L KCl, 0.2 g/L KH2PO4, 8 g/L NaCl, and 1.15 g/L Na2HPO4) containing 0.5 mM EDTA4Na and 3 mg/L Phenol Red.
Use:
Note: Washing or neutralizing of Accutase is not required in routine cell passaging.
General Dissociation:
This entire procedure should be done in a laminar flow hood using proper aseptic technique.
Dissociation of human ESCs grown in Serum Free Media on hESC-qualified Basement Membrane Extract
Dissociation of adherent human or rat neuronal stem cells grown in Serum Free Media on coated dishes
References
1. Efficient Propagation of Single Cells Accutase-Dissociated human Embryonic Stem Cells, Bajpai, et al, Journal of Molecular Reproduction and Development, 2007, DOI
10.1002/mrd:1-10.
2. Human Embryonic Stem Cell-derived Dopaminergic Neurons Reverse Functional Deficit in Parkinsonian Rats, Yang, et al, Stem Cells, 2007; 0: 2007-0494v1.
3. Oxygen Reduces Accumulation of Type IV Collagen in Endothelial Cell Subcellular Matrix via Oxidative Stress, T. Brevig, et al, Artificial Organs, Volume 30 Issue 12
Page 915-921, December 2006.
4. Canine hemangiosarcoma originates from hematopoietic precursors with potential for endothelial differentiation, Lamerota-Kozicki et al., Experimental Hematology, Vol. 34 Pages 870-878, April 2006.
5. The JAK3 inhibitor WHI-P154 prevents PDGF-evoked process outgrowth in human neural precursor cells, Richards et al., Journal of Neurochemistry, Vol. 97 Page 201,
April 2006.
6. Nuclear factor-ÊB controls the reaggregation of 3D neurosphere cultures in vitro, Widera et al., European Cells and Materials, Vol. 11, Pages 76-85, 2006.
7. Rescue Purification Maximizes the Use of Human Islet Preparations for Transplantation, Ichii et al., American Journal of Transplantation, Vol.
Once reconstituted, AccutaseLZ works just like Accutase. It is a cell detachment solution of proteolytic and collagenolytic enzymes. It is useful for the routine detachment of all cells including stem cells from standard tissue culture plastic-ware, adhesion coated plastic-ware, and polymers. Accutase performs exceptionally well in detaching cells for the analysis of cell surface markers, virus growth assay, quiescence assays by serum starvation, transformation assays by oncogene transfection, neural crest cell migration assays, cell proliferation, cell haptotaxis, tumor cell migration assays, routine cell passage, production scale-up (bioreactor), and flow cytometry.
Cell lines tested for Accutase application includes fibroblasts, keratinocytes, vascular endothelial cells, hepatocytes, vascular smooth muscle cells, hepatocyte progenitors, primary chick embryo neuronal cells, bone marrow stem cells, adherent CHO and BHK cells, macrophages, 293 cells, L929 cells, immortalized mouse testicular germ cells, MRC5, 3T3, Vero, COS, HeLa, NT2, MG63, M24 and A375 metastatic melanoma, gliomas U251, D54, HT1080 fibrosarcoma cells, Sf9 insect cells, human embryonic stem cells, human mesenchymal stem cells and human neural stem cells. Accutase does not contain mammalian or bacterial derived products.
Intended Use
Accutase is a direct replacement for trypsin cell detachment solution.
For research use only. CAUTION: Not intended for human or animal diagnostic or therapeutic uses. Contact us for cGMP version.
Reconstitution
- Place 400 mls of cold to room temp dH2O into a 500 ml graduated cylinder. Do not use warm or hot water. Accutase is temperature sensitive.
- Remove cap of 20X lyophilized Accutase vial and add 40 mls of cold dH20 to vial. Recap and vortex 1-2 minutes or invert and swirl until dissolved.
- Pipette the entire contents of the Accutase vial into the graduated cylinder. Use a pipette to insure complete transfer of contents.
- Rinse the vial twice with 25 ml of cold dH2O and add to graduated cylinder.
- Bring volume of graduated cylinder up to 500 ml with cold dH2O.
- Cover with parafilm and invert several times to mix.
- Aseptically filter the entire 500 mls through a 0.22μ filter. Use a low protein binding affinity filter such as cellulose acetate membrane.
- Accutase is stable when stored at 2 to 8 °C for 2 months.
Precautions
Do not store Accutase at room temperature. It is recommended to thaw Accutase at 4°C overnight or in a bath of cool water. Do not thaw at 37 °C.
Storage & Shelf Life
Store at -20C lyophilized, 2-8C defrosted. 12 month shelf life frozen.
Formulation:1X ACCUTASE enzymes in Dulbecco’s PBS (0.2 g/L KCl, 0.2 g/L KH2PO4, 8 g/L NaCl, and 1.15 g/L Na2HPO4) containing 0.5 mM EDTA4Na and 3 mg/L Phenol Red.
Use:
Note: Washing or neutralizing of Accutase is not required in routine cell passaging.
General Dissociation:
This entire procedure should be done in a laminar flow hood using proper aseptic technique.
- Carefully aspirate all of the media from the cell culture flask. (Rinsing with PBS is not necessary.)
- Immediately add enough Accutase to the flask to cover the cells. (Typically 2.5 to 5ml for a T25 flask depending upon confluency and density of the cell culture.)
- Set the flask aside at room temperature (RT) for 5 to 10 minutes up to a maximum of 1 hr. Check the flask frequently to see if the cells have rounded rather than merely shrunken or no longer appear “spidery” while remaining attached to the bottom of the flask.
- Once the cells have turned into “balls”, smack the flask against the palm of your hand to dislodge any “stickers”.
- Gently disperse the cells and take a sample of the cell suspension to determine the viable cell density.
- Add an aliquot of the detached cells to fresh media in new flasks. Place the flasks into the 37 °C incubator. No neutralization steps are required. The cells will reattach within a few minutes depending upon cell type.
Dissociation of human ESCs grown in Serum Free Media on hESC-qualified Basement Membrane Extract
- Aspirate the media from culture dish and wash with 4mL of DPBS (w/o calcium and magnesium).
- Aspirate DPBS and add 2mL of Accutase to culture dish.
- Incubate for 2 to 5 minutes at RT until individual single cells start to round up.
- Gently rinse to remove cells off of the plate’s surface.
- Transfer cell suspension to 15mL conical tube. Gently pipette up and down until cells are in a single cell suspension.
- Add 8 ml of media to rinse any remaining cells off of the dish’s surface and transfer to the conical tube from Step 5.
- Take a 20μl sample of the cell suspension to determine viable cell density.
- Centrifuge conical tube containing the cell suspension at 200g for 4 minutes.
- Aspirate supernatant, resuspend in fresh medium and plate on coated dish(s). Incubate at 37 °C in a humidified 5% CO2 incubator.
Dissociation of adherent human or rat neuronal stem cells grown in Serum Free Media on coated dishes
- Aspirate the media from the culture dish and wash with 4 ml of DPBS (w/o calcium and magnesium)
- Aspirate DPBS and add 2ml of Accutase to culture dish.
- Incubate for 2 to 5 minutes at RT until individual single cells start to round up.
- Gently rinse to remove cells off of the plate’s surface.
- Transfer cell suspension to 15 ml conical tube. Gently pipette up and down until cells are in a single cell suspension.
- Add 8 ml of media to rinse any remaining cells off of the dish’s surface and transfer to the conical tube from Step 5.
- Take a 20μl sample of the cell suspension to determine the viable cell density.
- Centrifuge conical tube containing the cell suspension at 200g for 4 minutes.
- Aspirate supernatant, resuspend in fresh media and plate on coated dish(s). Incubate at 37 °C in a humidified 5% CO2 incubator.
References
1. Efficient Propagation of Single Cells Accutase-Dissociated human Embryonic Stem Cells, Bajpai, et al, Journal of Molecular Reproduction and Development, 2007, DOI
10.1002/mrd:1-10.
2. Human Embryonic Stem Cell-derived Dopaminergic Neurons Reverse Functional Deficit in Parkinsonian Rats, Yang, et al, Stem Cells, 2007; 0: 2007-0494v1.
3. Oxygen Reduces Accumulation of Type IV Collagen in Endothelial Cell Subcellular Matrix via Oxidative Stress, T. Brevig, et al, Artificial Organs, Volume 30 Issue 12
Page 915-921, December 2006.
4. Canine hemangiosarcoma originates from hematopoietic precursors with potential for endothelial differentiation, Lamerota-Kozicki et al., Experimental Hematology, Vol. 34 Pages 870-878, April 2006.
5. The JAK3 inhibitor WHI-P154 prevents PDGF-evoked process outgrowth in human neural precursor cells, Richards et al., Journal of Neurochemistry, Vol. 97 Page 201,
April 2006.
6. Nuclear factor-ÊB controls the reaggregation of 3D neurosphere cultures in vitro, Widera et al., European Cells and Materials, Vol. 11, Pages 76-85, 2006.
7. Rescue Purification Maximizes the Use of Human Islet Preparations for Transplantation, Ichii et al., American Journal of Transplantation, Vol.
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