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| Product Name | Trypsin-EDTA Solution, 0.25%, with Phenol Red, Porcine Parvovirus Tested, 100 mL |
| Catalog No. | ATR-CCR-0041 |
| Description | 0.25% (w/v) porcine trypsin with 0.02% EDTA in Hanks' Balanced Salt Solution (HBSS) with phenol red pH indicator, formulated for routine detachment of adherent mammalian cells from tissue culture plastic. Trypsin is a serine protease that cleaves peptide bonds at the C-terminal side of lysine and arginine residues in extracellular matrix proteins (fibronectin, collagen, laminin) and cell adhesion molecules (integrins, cadherins), releasing cells from the culture surface and from each other. EDTA enhances trypsin activity by chelating Ca2+ and Mg2+ that are required for integrin-mediated and cadherin-mediated cell adhesion. Phenol red provides visual confirmation of pH (salmon-pink at pH 7.4, yellow at pH <6.5 indicating exhaustion). The solution is porcine parvovirus tested, mycoplasma tested, and sterile-filtered through 0.1 um membrane. This is the standard concentration for most adherent cell lines. |
| Intended Use | Enzymatic detachment of adherent mammalian cells from tissue culture plastic and dissociation of cell aggregates for: routine cell passaging; cell counting and viability assessment; harvesting cells for cryopreservation; preparing single-cell suspensions for flow cytometry; seeding cells for experiments (transfection, drug treatment, differentiation); and subculturing of established cell lines including HeLa, HEK293, MCF-7, HCT116, A549, Vero, MDCK, CHO, NIH/3T3, and many others. |
| Principle / Technology | Trypsin (EC 3.4.21.4) is a pancreatic serine protease with specificity for cleavage at the C-terminal side of lysine and arginine residues, unless followed by proline. In cell culture, trypsin digests the protein components of the extracellular matrix (fibronectin, collagen types I and IV, laminin) and cell surface adhesion proteins (integrin extracellular domains), causing cells to round up and detach from the culture surface. EDTA chelates Ca2+ (required for integrin ligand binding) and Mg2+, further weakening cell-matrix and cell-cell adhesions. The combination of trypsin and EDTA produces faster, more complete detachment with shorter exposure times compared to trypsin alone, reducing cellular damage. |
| Detection Method | 1) Pre-warm Trypsin-EDTA to 37 C; 2) Aspirate culture medium from cell monolayer; 3) Rinse cells with sterile PBS (without Ca2+/Mg2+) to remove residual serum (contains trypsin inhibitors); 4) Add enough Trypsin-EDTA to cover monolayer (approximately 0.5-1 mL per T25, 1-2 mL per T75 flask); 5) Incubate at 37 C for 2-10 min, monitoring under microscope for cell rounding; 6) Gently tap flask to dislodge cells; 7) Add complete medium (with serum — contains trypsin inhibitors) to inactivate trypsin; 8) Pipette gently to dissociate cell clumps; 9) Count and subculture as needed. |
| Sample Type | Adherent mammalian cell monolayers on tissue culture-treated plastic (T-flasks, dishes, multi-well plates). |
| Performance Range / Specifications | Trypsin concentration: 0.25% (2.5 g/L, approximately 100-150 BAEE U/mL); EDTA concentration: 0.02% (0.2 g/L, 0.54 mM); pH: 7.2-7.6; osmolality: 270-320 mOsm/kg; enzyme activity: 100-150 USP trypsin units/mL (or 1,000-1,500 BAEE units/mL); sterility: negative; mycoplasma: negative; porcine parvovirus: negative. |
| Sensitivity / LOD | Effective cell detachment typically achieved within 2-5 min at 37 C for most established cell lines; >95% detachment efficiency within 10 min for standard cell lines. |
| Specificity | Trypsin specifically cleaves at lysine and arginine residues (not followed by proline); does not degrade collagen if collagen is triple-helical (cleaves only denatured collagen/gelatin); EDTA non-specifically chelates Ca2+ and Mg2+, affecting all divalent cation-dependent adhesion systems. |
| Reaction Conditions / Protocol | Optimal activity at 37 C, pH 7.2-7.8; activity inhibited by: serum (contains alpha-1-antitrypsin and alpha-2-macroglobulin), soybean trypsin inhibitor (SBTI), PMSF, AEBSF, and leupeptin; EDTA activity neutralized by excess Ca2+ or Mg2+ in medium. |
| Components / Formulation | Trypsin-EDTA Solution, 0.25%, with phenol red: porcine trypsin (2.5 g/L), EDTA disodium salt (0.2 g/L), NaCl (8.0 g/L), KCl (0.4 g/L), glucose (1.0 g/L), NaHCO3 (0.35 g/L), phenol red (10 mg/L) in water for injection; 0.1 um sterile-filtered, 100 mL bottle. |
| Storage Conditions | Store at -20 C for long-term; store at 2-8 C after thawing, use within 2 weeks; avoid repeated freeze-thaw cycles (>3); protect from light. |
| Shelf Life | 24 months at -20 C; 2 weeks at 2-8 C after thawing; 24 months in frozen aliquots. |
| Package Specifications | 100 mL sterile PETG bottle; also available in 500 mL. |
| Product Form | Clear, salmon-pink sterile liquid (phenol red indicator); no particulates. |
| Quality Control | Each lot: trypsin activity (100-150 USP U/mL or 1,000-1,500 BAEE U/mL); pH 7.2-7.6; osmolality 270-320 mOsm/kg; sterility (negative, USP <71>); mycoplasma (negative); porcine parvovirus (negative, PCR); cell detachment test: HeLa cells detach >95% within 2 min at 37 C; cell viability after trypsinization: >90% by trypan blue exclusion. |
| Key Features | 0.25% trypsin with 0.02% EDTA — standard concentration; porcine parvovirus tested; phenol red pH indicator; sterile 0.1 um filtered; mycoplasma tested; HBSS-based formulation with glucose; effective for most adherent cell lines. |
| Purity | Trypsin: >90% pure by activity per mg protein (purified from porcine pancreas); EDTA: USP grade; all components: cell culture grade or USP. |
| Concentration | Trypsin: 0.25% (2.5 g/L); EDTA: 0.02% (0.2 g/L, 0.54 mM); enzyme activity: 100-150 USP U/mL. |
| Activity / Unit Definition | Trypsin specific activity: typically 6,000-10,000 BAEE U/mg protein; 0.25% solution has 100-150 USP U/mL (1,000-1,500 BAEE U/mL). |
| Molecular Weight | Porcine trypsin: ~23.8 kDa; EDTA disodium dihydrate: 372.24 g/mol. |
| Source / Origin | Trypsin: purified from porcine (pig) pancreas — animal derived; porcine parvovirus tested per regulatory requirements; EDTA: synthetic; all other components: synthetic or USP grade; manufactured in ISO-certified facility. |
| pH Range / Optimal pH | Optimal pH 7.2-7.8 for trypsin activity; phenol red indicator: salmon-pink at pH 7.4, yellow at pH <6.5; stable at pH 6.0-8.5. |
| Shipping Conditions | Frozen (-20 C or dry ice); must remain frozen upon receipt; upon arrival, store at -20 C. |
| Expiration Date / Stability | 24 months at -20 C; 2 weeks at 2-8 C; avoid >3 freeze-thaw cycles (trypsin autolysis reduces activity). |
| Regulatory / Compliance | For research use only; not for diagnostic or therapeutic use. Not for human or animal injection. Contains porcine-derived trypsin — some countries restrict import of animal-derived products. Porcine parvovirus tested for additional safety. Animal-derived product — handle per institutional biosafety guidelines. |
| Compatibility | Compatible with tissue culture-treated polystyrene (TC-treated plastic), glass, and most cell culture surfaces. Incompatible with untreated polystyrene (bacteriological plastic) — cells will not attach, making trypsinization irrelevant. For serum-free or low-serum culture, inactivate trypsin with soybean trypsin inhibitor (SBTI, 0.5 mg/mL in PBS) instead of serum-containing medium. Some highly adherent cell lines (MDCK, primary fibroblasts) may require longer trypsinization times (5-15 min). For very sensitive cells, use trypsin diluted 1:1 to 1:5 with PBS or use recombinant trypsin (TrypLE) for gentler dissociation. Trypsin-EDTA should not be used for: embryonic stem cell passaging (use mechanical or EDTA-only methods), neural stem cell neurospheres (use Accutase or mechanical dissociation), or cell lines grown in suspension. |
| Recommended Buffer System | HBSS-based: NaCl (137 mM), KCl (5.4 mM), Na2HPO4 (0.34 mM), KH2PO4 (0.44 mM), glucose (5.6 mM), NaHCO3 (4.2 mM), phenol red (10 mg/L); no Ca2+, no Mg2+. |
| Application Notes / Precautions | Pre-warm trypsin to 37 C before use. Do not leave trypsin in a 37 C water bath for extended periods (autolysis reduces activity). For each cell line, establish the minimum trypsinization time: monitor cells under microscope, and as soon as cells round up, gently tap flask and add medium. Over-trypsinization reduces viability and may cleave cell surface receptors. Serum-containing medium contains trypsin inhibitors (alpha-1-antitrypsin) that stop the reaction; always use at least 5% serum to neutralize. For cells that are difficult to trypsinize: pre-rinse with EDTA (0.02%) in PBS without trypsin for 5-10 min at 37 C to chelate cations before adding trypsin-EDTA. To prepare frozen aliquots: thaw bottle at 2-8 C overnight, aliquot aseptically, and immediately refreeze at -20 C. Do not store thawed trypsin-EDTA at RT. |
| Batch-to-Batch Consistency | Trypsin activity 100-150 USP U/mL; HeLa detachment >95% in 2 min; cell viability >90% after trypsinization; sterility and mycoplasma negative. |
For research use only, not for clinical use.
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