After transfection of the DCMECs as described above, the supernatant from the cultures was collected and the levels of the secreted β-casein, triglyceride and lactose were determined using the ELISA Kit for β-casein (CSN2) (New England Biolabs Inc., Beverly, MA, USA), Triglyceride (TG) GPO-POD Assay Kit (ApplyGEN) and Lactose & D-galactose (Rapid) Assay Kit (Megazyme, Bray, Ireland) according to the manufacturers' instructions. All experiments were performed in triplicate.
Lactose d galactose rapid assay kit
Manufactured by Megazyme
Sourced in Ireland
The Lactose & D-galactose (Rapid) Assay Kit is a laboratory equipment product designed for the rapid determination of lactose and D-galactose levels. The kit provides a method for the enzymatic measurement of these analytes.
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6 protocols using lactose d galactose rapid assay kit
1
Quantification of Milk Secretion Factors
2
Quantification of Milk Protein Secretion
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Following transfection, DCMECs were incubated with serum- and antibiotic-free medium for 36 h (Pten overexpression) or 48 h (siRNA inhibition of Pten). Culture medium were then collected using a glass dropper and transferred to one of the following detection kits (all used according to manufacturer’s instructions): ELISA Kit for Casein Beta (CSN2; New England Biolabs Inc.,Beverly, MA, USA); Triglyceride (TG) GPO-POD assay kit (Applygen Tech Inc.) and Lactose & D-galactose (Rapid) assay kit (Megazyme, Bray Business Park, Bray, Ireland). After DCMECs were treated with PRL and glucose for 24 h as described previously, culture medium were collected for detection using the a forementioned kits.
3
Milk Composition Analysis Protocol
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Milk lactose concentration was determined by Lactose/D- galactose (Rapid) Assay kit (Megazyme international Ireland Ltd., Wicklow, Ireland). Prolactin, progesterone, insulin (1μIU/mL = 1 ng/mL× 21.2), and IGF-1 content in milk infranatant were determined by radioimmunoassay (RIA) method using commercial kits (human, converted to pig estimates) (Tianjin Jiuding Medical and Biological Engineering Co., Ltd., Tianjin, China) as described by Foisnet et al. [49 (link)].
4
Quantifying Secreted Triglycerides and Lactose
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The amount of triglyceride and lactose secreted into the culture medium was determined using commercially available assay kits (triglyceride detection kit: ApplyGEN, Beijing, China; lactose assay kit: Lactose/d -galactose (Rapid) Assay Kit, Megazyme, Ireland, UK) according to the manufacturer’s recommended protocol [56 (link)].
5
Quantification of Secreted Milk Components
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At 48 h post-transfection, the culture supernatant was collected for detection. β-casein, lactose secretion, and triglycerides were detected using an ELISA Kit for Casein Beta (CSN2) (New England Biolabs Inc., Beverly, Massachusetts, USA), a Lactose & D-Galactose (Rapid) Assay Kit (Megazyme, Bray Business Park, Bray, Ireland), and a Triglyceride (TG) GPO-POD Assay Kit (Applygen Tech Inc., Beijing, China) according to the manufacturer’s instructions. All experiments were performed in triplicate.
6
Colostrum Nutrient Composition Analysis
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Nutrient composition of colostrum was determined from samples diluted 1/10, 1/100 or 1/1000 in PBS 1× as follows: lactose content was measured from 200 μl of diluted samples with the Lactose/d -Galactose (Rapid) Assay Kit (Megazyme, Wicklow, Ireland) according to the manufacturer's instructions; the protein content was measured from 100 μl of diluted samples with the Pierce™ BCA Protein Assay Kit (ThermoFisher, Rockford, IL, USA) and lipid content was analysed by hydrolysis according to an internal method adapted from NF ISO 6492 by the Artemis Laboratory (Janzé, France) from 1 ml of frozen raw colostrum.
Bioactive molecules were analysed as follows: lactoferrin concentration was measured from 50 μl of diluted samples through the Sheep Lactoferrin (LF) ELISA Kit (Mybiosource, San Diego, CA, USA) according to the manufacturer's recommendation; the IgG concentration was analysed by radial immunodiffusion by CIAL Sud Ouest laboratory (Auch, France) from 1 ml of raw colostrum and 1 ml of a lamb blood sample. For biochemical analyses, no technical replicate was performed due to volume limitation, but all biological replicates were analysed (i.e. from six to fourteen animals per group).
Bioactive molecules were analysed as follows: lactoferrin concentration was measured from 50 μl of diluted samples through the Sheep Lactoferrin (LF) ELISA Kit (Mybiosource, San Diego, CA, USA) according to the manufacturer's recommendation; the IgG concentration was analysed by radial immunodiffusion by CIAL Sud Ouest laboratory (Auch, France) from 1 ml of raw colostrum and 1 ml of a lamb blood sample. For biochemical analyses, no technical replicate was performed due to volume limitation, but all biological replicates were analysed (i.e. from six to fourteen animals per group).
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