Mice were housed under a 12 h light/dark cycle with free access to food and water. FVB/NJ (Stock# 001800) mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA) and used as wildtype. Pregnant FVB females, 3–8 months of age, were euthanized at E11.5, the uterus was dissected and embryos were taken out and placed into 1x PBS (Gibco, 14190–250). Embryos were individually collected in either TRIzol (Invitrogen, 15596) and lysed by pipetting for total RNA isolation or collected in 2 ml safe-lock tubes (Eppendorf) in 1x PBS, supernatant was removed and embryos were snap frozen in liquid nitrogen. For lysates, embryo pellets were homogenized by cryo-milling after addition of a 2.5 or 5 mm steel bead using a tissue lyser (QIAgen TissueLyser II) at 25 Hz for 15 seconds 3–6 times, and the powder was either processed directly or snap frozen in liquid nitrogen and stored at −80°C. All animal work was performed in accordance with protocols approved by Stanford University’s Administrative Panel on Laboratory Animal Care.
Phosphate buffered saline (pbs)
Manufactured by Eppendorf
Phosphate-buffered saline (PBS) is a common buffer solution used in various laboratory applications. It is a balanced salt solution that maintains a physiological pH and osmolarity, making it suitable for preserving and suspending biological samples. PBS is primarily composed of sodium chloride, potassium chloride, sodium phosphate, and potassium phosphate.
Automatically generated - may contain errors
Lab products found in correlation
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (4 mentions)
Fvb nj stock 001800 mice, by Jackson ImmunoResearch (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Tissuelyser 2, by Qiagen (2 mentions)
Safe lock tube, by Eppendorf (2 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
5 protocols using phosphate buffered saline (pbs)
1
Isolation and Cryopreservation of Mouse Embryos
2
Isolation of Mouse Embryonic Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mice were housed under a 12 h light/dark cycle with free access to food and water. FVB/NJ (Stock# 001800) mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA) and used as wildtype. Pregnant FVB females, 3-8 months of age, were euthanized at E11.5, the uterus was dissected and embryos were taken out and placed into 1x PBS (Gibco, 14190-250) . Embryos were individually collected in either TRIzol (Invitrogen, 15596) and lysed by pipetting for total RNA isolation or collected in 2 ml safe-lock tubes (Eppendorf) in 1x PBS, supernatant was removed and embryos were snap frozen in liquid nitrogen. For lysates, embryo pellets were homogenized by cryo-milling after addition of a 2.5 or 5 mm steel bead using a tissue lyser (QIAgen TissueLyser II) at 25 Hz for 15 seconds 3-6 times, and the powder was either processed directly or snap frozen in liquid nitrogen and stored at -80°C. All animal work was performed in accordance with protocols approved by Stanford University's Administrative Panel on Laboratory Animal Care.
3
Immunophenotyping of PDLSCs by Flow Cytometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Flow cytometry was performed according to the manufacturer's instructions (BD Biosciences, San Jose, CA, USA) to detect the immunophenotype of PDLSCs. Briefly, 1 × 106 hPDLSCs were washed 3 times with PBS (Thermo Fisher Scientific, Waltham, MA, USA). Then, cells were resuspended with 500 µL of PBS and aliquoted into six 1.5 mL Eppendorf (EP) tubes. MSC-positive markers (Stro1-PE, CD105-PE, and CD29-PE) and MSC-negative markers (CD34-PE and CD45-PE) antibodies were then added to each tube, respectively. After 30 min incubation in the dark at 37°C, unbound antibody was washed away with PBS and detected by flow cytometry.
4
Nanoparticle Stability and Hemolysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The particles size of prepared NPs (dissolved in water) was detected by DLS in different time points with stored at RT. Meanwhile, the same process was performed after the NPs dispersed in PBS, normal saline (NS), DMEM, DMEM+10% FBS. In addition, the particles size of NPs dispersed in mice plasma at different time points were also tested to investigate the stability of NPs in physiological environment.
The fresh blood was obtained from healthy mice to isolate the red blood cells (RBCs), with centrifugation at 2500×g for 5 min and washed by PBS for three times (Eppendorf). The 2% RBCs re-suspension was prepared with PBS, and 0.5 mL suspension incubated with 0.5 mL NPs (dissolved in PBS) at 37 °C for 3 h, followed by centrifugation at 2500×g for 5 min (Shimadzu). The OD value of different groups was tested at 504 nm, and the hemolysis ratio was measured compared to the negative control of PBS and positive control of water. More than 5% indicated a hemolytic effect.
The fresh blood was obtained from healthy mice to isolate the red blood cells (RBCs), with centrifugation at 2500×g for 5 min and washed by PBS for three times (Eppendorf). The 2% RBCs re-suspension was prepared with PBS, and 0.5 mL suspension incubated with 0.5 mL NPs (dissolved in PBS) at 37 °C for 3 h, followed by centrifugation at 2500×g for 5 min (Shimadzu). The OD value of different groups was tested at 504 nm, and the hemolysis ratio was measured compared to the negative control of PBS and positive control of water. More than 5% indicated a hemolytic effect.
5
Saponin-Chelex DNA Extraction from DBS
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DNA extraction was done using the Saponin-Chelex extraction method. Saponin (0.5%) in phosphate buffered saline (PBS) was prepared by dissolving 1 g of saponin and one tablet of PBS (Sigma-Aldrich, USA) in 200 ml of sterile distilled water, shaken vigorously and allowed to stand till the saponin and PBS dissolved completely. Each of the dried blood spot was cut out from the DBS papers with sterilized scissors with serrated edges and each of the discs were transferred to pre-labelled Eppendorf tubes. 1 ml of the saponin-PBS solution was added to the specimen in the Eppendorf tubes and allowed to stand for 24 h at 4 °C for complete haemolysis.
The specimens were washed three times in PBS with intermittent centrifugation at 13,000 rpm for 5 min after each wash. Subsequently, 1 ml of PBS was added to the sediment, and then it was vortexed for 1 min and incubated for 30 min at 4 °C. It was centrifuged again for 5 min at 13,000 rpm and the supernatant decanted.
Fifty microlitres of freshly prepared Chelex solution was added to the tubes followed by 150 μl of distilled water to obtain 5% of the Chelex solution. It was then vortexed and incubated in a block heater set at 99 °C for 20 min, centrifuged at 13,000 rpm for 5 min and the supernatants decanted into newly labelled Eppendorf tubes. The supernatants contain the DNA.
The specimens were washed three times in PBS with intermittent centrifugation at 13,000 rpm for 5 min after each wash. Subsequently, 1 ml of PBS was added to the sediment, and then it was vortexed for 1 min and incubated for 30 min at 4 °C. It was centrifuged again for 5 min at 13,000 rpm and the supernatant decanted.
Fifty microlitres of freshly prepared Chelex solution was added to the tubes followed by 150 μl of distilled water to obtain 5% of the Chelex solution. It was then vortexed and incubated in a block heater set at 99 °C for 20 min, centrifuged at 13,000 rpm for 5 min and the supernatants decanted into newly labelled Eppendorf tubes. The supernatants contain the DNA.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!