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Stromal vascular fraction cells were isolated following established protocols [14].
Accordingly, a mean of 4.8 × 107 stromal vascular fraction cells, which contained a mean of 4.0 × 106 stem cells, were used for MSC injection.
Thus, after isolation, the adipose-derived stem cells represented a mean of 8.7%% of the stromal vascular fraction cells (range, 6.9 to 11.2 %).
To evaluate the frequency of mesenchymal-like progenitors in the stromal vascular fraction, cells were cultured in T-25 flasks at a final concentration of 16 cells/cm2.
After the stromal vascular fractions were isolated, a mean of 4.0 × 106 stem cells (8.7%% of 4.6 × 107 stromal vascular fraction cells; range, 3.2 to 5.2 × 106 cells) were prepared.
For isolation of the nuclear fraction, cells were lysed using the NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo Scientific, Rockford, USA).
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Bark INA was located mostly in the cell wall fraction (cell walls and intercellular structural components).
The analysis in paired samples (CD19+ fraction cell and non-CD19+ fraction cell) showed that the presence of rs2307842 and IGHV unmutated genes determined HSP90B1 overexpression in B lymphocytes from CLL patients.
To separate nuclear and cytoplasmic fractions, cells were fractionated with NE-PER NuCytoplasmicytoplasmic Extraction Reagent (Pierce Biotechnology, Rockford, IL, USA).
No engraftment was observed when NOG mice were transplanted with either the Lin−CD34+CD38−CD90+CD45RA− cell fraction (385 3000 cells injected) or the more differentiated Lin−CD34+CD38−CD90−CD45RA− cell fraction (150 1800 cells injected).
For nuclear fractions, cells were scraped with cold soft-lysis buffer (50 mM Tris-HCl, 10 mM EDTA, 0.1%NP-40NP-40d 10% glycerol) supplemented with protease inhibitors.
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