Journal: bioRxiv

Article Title: Characterization of CRISPR/Cas9 RANKL knockout mesenchymal stem cell clones based on single-cell printing technology and emulsion coupling assay as a low-cellularity workflow for single-cell cloning

doi: 10.1101/2020.08.17.253559

Figure Lengend Snippet: hTERT MSCs were transfected with either pmaxGFP (transfection efficiency 61%) or pNV-RANKL/KO (transfection efficiency 6%). Two days post-transfection, GFP expressing single-cells were isolated by SCP (see details below). In (A) the SCP (f.sight™) cartridge ejection area is recorded during the printing process, showing a fluorescent single-cell before (01-03), at (04), and after (05) ejection. The SCP algorithm intercepts and deflects cells that are not qualifying the preset parameters (size threshold was set from 15 – 30 μ m, roundness from 0.6 – 1, where 1 reflects the perfectly circular object). In (B) and (D) the roundness is plotted against cell diameter and in (C) and (E) the relative fluorescence intensity unit (RFU) is plotted against cell diameter - all detected events are in gray. The red dots represent printed single-cells qualified to the preset parameters (see below). The dotted orange line represents the thresholds of parameters for selecting single cells. (F) The printed single-cells were evaluated for clonality (n= 451 transfected printed cells). The recorded image series of each qualified printing process was evaluated manually, categorizing single, multiple-cell, uncertain, or void printing events. (G) After 2 weeks colonies obtained from single cells were counted and thereby the cloning efficiency determined (n=227 transfected printed cells, n=650 non-transfected printed cells). The single-cell printing parameters applied for pmaxGFP transfected hTERT MSCs: laser intensity of 20 - 40%, an exposure time of 15 - 30 ms and a fluorescence threshold of 30 - 250 RFU while for pNV-RANKL/KO transfected hTERT MSCs laser intensity of 90-95%, exposure time of 80 - 120 ms and a fluorescence threshold of 50 - 250 RFU.

Article Snippet: Three vectors were purchased with the following gRNA sequences: pNV-RANKL1/KO - 5’-CAGGAATTACAACATATCGT-3’ (472221110290), pNV-RANKL2/KO - 5’-CAGCGATGGTGGATGGCTCA-3’ (472221110390), pNV-RANKL3/KO - 5’-TTAATAGTGAGATGAGCAAA-3’ (472221110490). pmaxGFP (purchased from Lonza, Human MSC Nucleofector ® Kit) was used to control transfection efficiency.

Techniques: Transfection, Expressing, Isolation, Fluorescence, Clone Assay

Journal: Advances in Wound Care

Article Title: A Small Chimeric Fibronectin Fragment Accelerates Dermal Wound Repair in Diabetic Mice

doi: 10.1089/wound.2015.0666

Figure Lengend Snippet: FNIII1HRGD supports MSC adhesion and proliferation. Tissue culture plates were precoated with increasing concentrations of FNIII1HRGD (50–400 nM). MSCs were seeded (1 × 104 cells/cm2) onto protein-coated plates. (A) Phase contrast microscopy images were taken at 72 h postseeding. Images represent one of three experiments performed. Scale bar, 50 μm. (B) Cell number was determined at 1, 4, and 7 days postseeding using crystal violet, as described in the Materials and Methods section. Data are presented as mean absorbance ± SEM of three experiments performed in triplicate. *Significantly different from FNIII1HRGD at corresponding time point, p < 0.05 (ANOVA). MSC, mesenchymal stem cell.

Article Snippet: 21 Human mesenchymal stem cells (MSCs) and MSC Growth Medium were from Lonza.

Techniques: Microscopy