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Cells that migrated out of the tail pieces were transferred to new plates (Passage 2) and maintained in DMEM containing 10% FBS.
Pieces were transferred into 30 ml of aCSF containing 1.3 mg/ml trypsin, 0.67 mg/ml hyaluronidase, and 0.2 mg/ml kynurenic acid (all from Sigma) and incubated, under continuous oxygenation and stirring, for 90 min at 32 34°C.
For HDF cultures established by enzymatic dissociation of the skin, dermal pieces were transferred to a solution of Collagenase I (Worthington Biochemical) at 200 units/mL of 1× PBS including 0.3 mM CaCl2.
The amount of meat transferred from one individual to another was estimated based on body-part weight (obtained from the literature, in cases when a complete body part was transferred, [34] [36]), relation of meat transferred to chimpanzee hand size (in cases when single pieces were transferred; a 5×5 cm piece of tissue from a red colobus weighed 50 g, K. Mätz-Rensing pers. comm).
Every few weeks until harvest, callus pieces were transferred to new plates containing the same media.
Two pieces were transferred to opposite sides of a rectangular plastic mould (75 × 25 × 15 mm).
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The pieces are transferred to a 50 ml conical tube and rinsed 3 times with 2 ml of ice-cold RPMI-1640 contantibioticsbiotics, 3% fetal bovine serum (FBS) and 20 mM HEPES, followed by decanting of the supernatant.
Briefly, cells were developed on agar for 48 h and the whole agar piece was transferred to 50 ml tubes containing 10 ml phosphate buffer (PB: 5 mM Na2HPO4 and 5 mM KH2PO4, pH 6.2).
After the incubation, the liquid from the gel piece was transferred to a new labeled tube.
An individual stem piece was transferred to a Leica model CM3050 cryostat (Leica Microsystems, Germany) and allowed to equilibrate in the cryostat chamber (chamber temperature -15°C and object temperature -25°C).
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