Wild type and two-timepoint edited 23–25 dpf zebrafish brains were similarly processed for inDrops single-cell transcriptome barcoding4 (link),53 (link) except that two-timepoint edited zebrafish were first heat shocked for 45 min at 37 C to induce scGESTALT barcode mRNA expression. Whole brains were dissected and dissociated using the Papain Dissociation Kit (Worthington), according to the manufacturer’s instructions with the following modifications to ensure high quality cell isolation for scRNA-seq54 . Brains were dissociated with 900 μl of 10 units/ml of papain in Neurobasal media (Life Technologies) and incubated at 34 C for 20–25 min with gentle agitation. Samples were then gently triturated with p1000 and p200 tips until large pieces of tissues were no longer visible. Dissociated cells were washed 2x with DPBS (Life Technologies) at 4 C and sequentially filtered through 35 μm (BD Falcon) and 20 μm (Sysmex) mesh filters. Cells were resuspended in 300–400 μl DPBS and counted using an automated Bio-Rad counter. Cells were then diluted to ~100,000 cells/ml in 18% optiprep/DPBS solution. Cells were loaded onto the inDrops device and encapsulated at a rate of 10,000–20,000 per hour. Transcriptomes were obtained for ~70% of cells introduced into the device.
Dulbecco phosphate buffered saline (dpbs)
Manufactured by Bio-Rad
Sourced in United States
DPBS is a sterile, isotonic phosphate-buffered saline solution commonly used as a buffer and washing agent in various cell culture and molecular biology applications. It maintains a physiological pH and osmolarity, helping to preserve the viability and integrity of biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Dulbecco phosphate buffered saline (dpbs), by Thermo Fisher Scientific (3 mentions)
Neurobasal media, by Thermo Fisher Scientific (2 mentions)
Papain dissociation kit, by Worthington (2 mentions)
Dulbecco phosphate buffered saline (dpbs), by Merck Group (1 mentions)
Tali image based cytometer, by Thermo Fisher Scientific (1 mentions)
Fcs express research edition software, by De Novo Software (1 mentions)
Tali cell cycle solution, by Thermo Fisher Scientific (1 mentions)
3 protocols using dulbecco phosphate buffered saline (dpbs)
1
Single-cell transcriptomics of zebrafish brains
2
Single-cell transcriptomics of zebrafish brains
3
Cell Cycle Analysis of C2C12 Myoblasts
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Cell cycle analysis was performed as in Pierucci et al. [45 (link)]. Briefly, C2C12 myoblasts were collected, washed once with DPBS (Sigma Aldrich, Milan, Italy) and fixed with ice-cold 70% ethanol in dH2O (Sigma Aldrich, Milan, Italy) to a concentration of 1 × 106 to −5 × 106 cells/mL. The samples were placed at least for a night at –20 °C, then ethanol was removed, and cells were washed with DPBS + 1%BSA (Bio-Rad, Hercules, CA, USA). After DPBS was removed, cells were stained with propidium iodide incubating them at room temperature for 30 min in the dark with the Tali®® Cell Cycle Solution (Life Technologies, Eugene, OR, USA) to a concentration of 1 × 105 to –5 × 106 cells/mL. Cell suspension (25 µL) was transferred in the dedicated slide and cell cycle analysis was performed using the Tali®® Image-Based Cytometer (Thermo Fisher Scientific, Carlsbad, CA, USA). The percentage of cells in each phase of the cell cycle was determined using FCS Express Research Edition software (version 4.03; De Novo Software, Morristown, NJ, USA).
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