Immediately following either MACS or FACS, NPCs were seeded at a density of 1x 105 cells per well in a 96 well plate to assay survival and stress at 24 hours after sorting. After 24 hours, media supernatant was collected for a colorimetric LDH assay (Abcam), which was carried out according to manufacturer’s guidelines and measured at 450nm with a 650nm reference on a Varioskan microplate reader. The remaining live cells were labelled with CytoCalcein violet (Abcam) and immediately imaged on a Leica DMIL LED Inverted Routine Fluorescence Microscope with a 5x and 20x objective. NPCs that had not undergone any sorting or manipulation other than regular passaging were also seeded for comparison. Additional NPCs were treated with 1μM Staurosporine (STS) for 1, 6 and 24 hours as a positive control for toxicity and cell death. The percentage of total well surface area covered by CytoCalcein violet-stained cells was determined by converting images to high contrast greyscale and submitting them for automatic thresholding and measurement in ImageJ.
Colorimetric ldh assay
Manufactured by Abcam
Sourced in United Kingdom
The Colorimetric LDH Assay is a laboratory equipment product that measures the activity of lactate dehydrogenase (LDH) in samples. LDH is an enzyme found in various cell types and is released upon cell damage or death. The assay quantifies LDH levels using a colorimetric reaction, providing a way to assess cellular cytotoxicity or tissue damage.
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2 protocols using colorimetric ldh assay
1
Analyzing NPC Survival and Stress Post-Sorting
2
Measuring Cell Viability and Injury
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The percentage of damaged or injured cells in our samples was measured indirectly by colorimetric LDH assay (Abcam, Cambridge, UK). For this purpose, the supernatants were collected on days 10, 14, 21 and 28 and processed according to the manufacturer's protocol for determining LDH activity. The absorbance intensity was measured at 490 nm by SpectraMax, and enzymatic activity was presented as mU/mL. In addition, a two-color fluorescence (calcein AM and ethidium homodimer; Invitrogen, Karlsruhe, Germany) cell viability assay was used for the simultaneous determination of live (green) and dead (red) cells; data were analyzed by two-photon microscopy.
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