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We consider the offline sorting buffer problem.
10 cells from single cell suspensions were suspended in 100 μL fluorescence-activated cell sorting buffer, and Fc receptor was blocked with anti-mouse CD16/32 (clone 93, Biolegend).
Antibodies include ESA-FITC (Dako, Cambridge, UK, BerEP4, F0860), CD44-APC (BD Pharmingen, Oxford, UK, 559942) and CD24-PE (Beckman Coulter, High Wycombe, UK, IMI1428U) For analysis, cells were resuspended in 500 μl of sorting buffer and passed through a 40 μm sieve.
Following 30 min on ice, the cells were washed in cold sorting buffer (phosphate-buffered saline supplemented with 2% FCS and 1% bovine serum albumin) and resuspended in sorting buffer containing 0.5 μg/ml of streptavidin PE-Cy5.
We are given a random-access sorting buffer with a limited capacity.
Cells were resuspended in sorting buffer (1 mM EDTA: 25 mM HEPES: 1% FCS: 10 µg/ml PI: PBS).
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The sorting buffers problem is motivated by many applications in manufacturing processes and computer science, among them car-painting and file servers architecture.
The yield of sorted nuclei in sort buffer (PBS) was determined with a hemacytometer, and 50 200 K nuclei were used for each ChIP.
Trypsinisation was stopped by the addition of medium, cells were spun down, washed in PBS and resuspended in 500 µl of sort buffer (1×PBS, 1mM EDTA, 25mM Hepes pH 7.0, 1% FCS).
For MNase fragmentation, nuclei in sort buffer were supplemented with PBS containing 0.1% Triton-X100 (PBS-Tx) to a volume of 400 µl, and CaCl2 to 1 mM.
For bath sonication, the nuclear suspension in sort buffer was adjusted to ChIP buffer conditions (10 mM Tris pH8, 100 mM NaCl, 0.1% sodium deoxycholate, 0.5% sarkosyl, 1% Triton-X100) in a volume of 500 µl.
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