Cold pbs

Manufactured by Thermo Fisher Scientific

Cold PBS is a sterile, isotonic phosphate-buffered saline solution used for maintaining the pH and osmolarity of cell cultures and other biological samples during handling and storage. It is pre-chilled to a low temperature to help preserve the integrity of temperature-sensitive samples.

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Lab products found in correlation

Paraformaldehyde (pfa), by Merck Group (2 mentions) Xylazine, by Bayer (2 mentions) Ketamine, by Pfizer (2 mentions) 10 cm dish, by BD (1 mentions) 8 well chamber slide, by Merck Group (1 mentions) 12 well plate, by Corning (1 mentions) Nucleofector 2b device, by Lonza (1 mentions) Amaxa mouse macrophage nucleofector kit, by Lonza (1 mentions) Inveon research workplace, by Siemens (1 mentions) Inveon mm system, by Siemens (1 mentions) Dmem medium, by Thermo Fisher Scientific (1 mentions) Rbc lysis buffer, by Thermo Fisher Scientific (1 mentions) 100 μm cell strainer, by Thermo Fisher Scientific (1 mentions) Sb202190, by Merck Group (1 mentions) Noggin, by R&D Systems (1 mentions) Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (1 mentions) R spondin 1, by R&D Systems (1 mentions) Tryple express, by Thermo Fisher Scientific (1 mentions) A83 01, by Bio-Techne (1 mentions) Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

Ice cold PBS (2 citations)

7 protocols using cold pbs

Macrophage Transfection Procedure

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Frozen differentiated BMDMs were recovered and plated at a density of 5 × 10⁶ cells in a 10 cm dish (Falcon) in RPMI medium containing 10% (vol/vol) FBS and 10% (vol/vol) L‐cell conditioned supernatant. The next day, cells were lifted in cold PBS (Gibco) and resuspended in RPMI medium containing FBS (Swanson & Isberg, 1995). Resuspended cells were aliquoted into 1.5 ml microfuge tubes at 1 × 10⁶ cells per tube and pelleted by centrifugations for 10 min at 200g. Cell pellets were resuspended in nucleofector buffer (Amaxa Mouse Macrophage Nucleofector Kit [Lonza]) and 2 μg of siRNA was added to each tube (Dharmacon). Cells were transferred to a cuvette and nucleofected in the Nucleofector 2b Device (Lonza) under Y‐001 program settings. Nucleofected macrophages were immediately recovered in RPMI medium containing 10% FBS and 10% L‐cell supernatant. Cells were plated in 8‐well chamber slides (Millipore Sigma) for microscopy assays or in 12‐well plates (Corning) to prepare extracts for immunoblotting.

Anand I.S., Choi W, & Isberg R.R. (2020). Components of the endocytic and recycling trafficking pathways interfere with the integrity of the Legionella‐containing vacuole. Cellular Microbiology, 22(4), e13151.

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Microstructural Analysis of Mouse Femurs

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Mouse femurs were harvested and all the connected tissues were removed, and the remaining tissue were stored in cold PBS (Gibco). The femurs were scanned with a Inveon MM system (Siemens, Munich, Germany). Briefly, the specimens were scanned at a voltage of 80 kV, a current of 450 μA, and an exposure time of 40 min in each of the 360 rotation steps. Three-dimensional reconstruction was performed by two-dimensional images. Bone mineral density (BMD), bone tissue ratio (BV/TV), bone surface area to volume ratio (BS/BV), bone trabecular number (Tb. N), bone trabecular thickness (Tb. Th), bone trabecular separation (Tb. Sp), and bone trabecular pattern factor (Tb. Pf) were calculated by Inveon Research Workplace (Siemens).

Xu Z., Yu Z., Chen M., Zhang M., Chen R., Yu H., Lin Y., Wang D., Li S., Huang L., Li Y., Yuan J, & Yin P. (2022). Mechanisms of estrogen deficiency-induced osteoporosis based on transcriptome and DNA methylation. Frontiers in Cell and Developmental Biology, 10, 1011725.

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Isolation of Fetal Liver and Bone Cells

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Tissues were kept in cold DMEM medium (Invitrogen) until dissection and processed on the same day of collection. Single-cell suspensions were generated from matched foetal liver and bone tissues after rinsing them with cold PBS (Gibco). Liver samples were passed through a 70 μm strain into a falcon tube prefilled with cold PBS. Bone marrow from long bones was isolated by flushing cold PBS into the diaphysis and collected into a falcon tube. Bone marrow from hip bone was collected by dissecting the bone with a sterile scalpel and flushing cold PBS in the marrow cavity into a falcon tube. The suspension obtained from long bones and hip bones was then passed through a 70 μm strain into a new falcon tube. Cells were then centrifuged for 5 minutes at 300 g, 4°C and the pellet was resuspended into the RBC lysis buffer (eBioscience) for 2 minutes at room temperature, after which 20 ml of cold PBS were added to stop the lysis reaction. RBC step was not performed when sorting erythroid cells. Live cell enrichment was performed using MACS columns (Miltenyi Biotec - 130-090-101) following the manufacturer's instructions. When sorting CD34+ or CD45+ cells, column enrichment was performed using MACS columns (Miltenyi Biotec -130-046-702 and 130-045-801 respectively for CD34+ and CD45+ cells), following the manufacturer's instructions.

Ranzoni A.M., Tangherloni A., Berest I., Riva S.G., Myers B., Strzelecka P.M., Xu J., Panada E., Mohorianu I., Zaugg J.B, & Cvejic A. (2021). Integrative Single-Cell RNA-Seq and ATAC-Seq Analysis of Human Developmental Hematopoiesis. Cell Stem Cell, 28(3), 472-487.e7.

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Establishing Human Intestinal Organoids

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Human enteroids were generated as previously described^{12 (link),29 (link)}. Briefly, engraftments were harvested and opened and pinned with mucosa facing upwards and then rinsed in cold PBS (Gibco). The tissues were then transferred to 2 mM EDTA (EDTA; Sigma-Aldrich) in PBS and rocked for 30 min on ice. After EDTA chelation, tissues were again washed in cold PBS, and crypts were manually removed from underlying submucosa and then filtered through a 100-μm cell strainer (Fisher Scientific). Crypts were then pelleted and resuspended in Matrigel (BD Biosciences) and overlaid with intestinal growth medium (Advanced DMEM/F-12, N2, B27, 15 mM HEPES, 2 mM L-glutamine, penicillin-streptomycin) supplemented with EGF (50 ng mL⁻¹), Noggin (100 ng mL⁻¹), R-spondin 1 (1 μg mL⁻¹) (R&D Systems), 50% Wnt3a conditioned medium, 1 mM N-acetylcysteine, 10 nM (Leu15)-Gastrin, 10 mM Nicotidamide, 10 μM SB202190 (Sigma-Aldrich), 500 nM A-83-01 (Tocris). 2.5 μM Thiazovivin and 2.5 μM CHIR99021 (Stemgent) were added to the medium for the first 2 d. Medium was changed every 3 d. Established enteroids were passaged over time by enzymatic (TrypLE Express, Life Technologies) and mechanical dissociation through an 18-gauge needle after 7 d in culture. Dissociated enteroids were then resuspended in PBS and pelleted before resuspension in Matrigel and culture conditions as above.

Watson C.L., Mahe M.M., Múnera J., Howell J.C., Sundaram N., Poling H.M., Schweitzer J.I., Vallance J.E., Mayhew C.N., Sun Y., Grabowski G., Finkbeiner S.R., Spence J.R., Shroyer N.F., Wells J.M, & Helmrath M.A. (2014). An in vivo model of human small intestine using pluripotent stem cells. Nature medicine, 20(11), 1310-1314.

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Rodent Euthanasia and Tissue Perfusion

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All experimental animal procedures were approved by the local animal care committee in Recklinghausen and conducted in compliance with federal and state guidelines for animal experiments in Germany (Permit Number: 84-02.04.2012.A300). Rats were maintained on a 12 hour light/dark cycle with ad libitum access to food and water. Rats were killed either by inhalation of CO₂ or intraperitoneal application of ketamine (60–80 mg/kg; Pfizer) and xylazine (10–15 mg/kg; Bayer) and perfused through the heart with cold PBS (Gibco) followed by paraformaldehyde (Sigma) (4% PFA in PBS).

Levin E., Leibinger M., Andreadaki A, & Fischer D. (2014). Neuronal Expression of Muscle LIM Protein in Postnatal Retinae of Rodents. PLoS ONE, 9(6), e100756.

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Rat Tissue Harvesting for Cell/Molecular Analysis

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All experimental procedures were approved by the local animal care committee in Recklinghausen (LANUV, Germany) and conducted in compliance with federal and state guidelines for animal experiments in Germany (approval number: 84-02.04.2015.A290). Rats were maintained on a 12-hour light/dark cycle with ad libitum access to food and water. In total 24 rats were used. Animals were killed either by inhalation of CO₂ for preparations of cell cultures, Western blot lysates and mRNA isolations or by intraperitoneal application of ketamine (60–80 mg/kg; Pfizer) and xylazine (10–15 mg/kg; Bayer) and perfusion through the heart with cold PBS (Gibco) followed by paraformaldehyde (Sigma) (4% PFA in PBS) for immunohistochemical analyses.

Levin E., Andreadaki A., Gobrecht P., Bosse F, & Fischer D. (2017). Nociceptive DRG neurons express muscle lim protein upon axonal injury. Scientific Reports, 7, 643.

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Lung Dissociation and Fixation

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Mice were anesthetized via IP Ketamine + Xylazine, followed by euthanasia via cervical dislocation and thoracotomy. The chest cavity was opened to expose the heart and lungs. The right ventricle was perfused with 5–10 mL of cold PBS (Gibco, 10010-023) to clear blood from the lungs. For tissue dissociation for organoids, the lungs were removed and placed in cold PBS on ice. For tissue fixation for histology and immunofluorescence, the trachea was cannulated and lungs were inflated to a pressure of 30 cm H₂O using 4% paraformaldehyde (PFA). Inflated lungs were immersed in a conical of 4% PFA and then left on a rocker at 4 °C overnight.

Toth A., Kannan P., Snowball J., Kofron M., Wayman J.A., Bridges J.P., Miraldi E.R., Swarr D, & Zacharias W.J. (2023). Alveolar epithelial progenitor cells require Nkx2-1 to maintain progenitor-specific epigenomic state during lung homeostasis and regeneration. Nature Communications, 14, 8452.

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