HBSS Balanced Salt Solution, Ca/Mg-Free, No Phenol Red

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• HBSS (Hank's Balanced Salt Solution)HBSS (Hank's Balanced Salt Solution) is an isotonic balanced salt solution used to wash cells, transport tissue, dilute reagents and hold cells briefly outside their growth medium. It contains 8.0 g/L sodium chloride, 1.0 g/L D-glucose, phosphate and 350 mg/L sodium bicarbonate, and is supplied either with calcium and magnesium (1.26 mM Ca, ~0.9 mM Mg total) or without them. Its low bicarbonate means it is designed for use at atmospheric CO2 or in sealed vessels, not for prolonged culture in a 5% CO2 incubator.
• Trypan Blue and the Dye Exclusion Viability AssayTrypan blue is a diazo dye used at 0.4% (w/v) to separate live from dead cells by dye exclusion: an intact plasma membrane keeps the charged dye out, so viable cells stay clear and refractile, while cells with a damaged membrane take the dye up and stain blue. In practice the cell suspension is mixed 1:1 with 0.4% trypan blue, loaded into a hemocytometer or an automated cell counter, and counted within 3-5 minutes to give both a cell concentration and a percent viability. Because it reports membrane integrity only, trypan blue counts cells that have already died -- it does not detect apoptotic or metabolically failing cells that still have an intact membrane, so it reads high compared with metabolic or flow-cytometry viability assays.
• ACK Lysis Buffer: Recipe, Composition and RBC Lysis ProtocolACK (Ammonium-Chloride-Potassium) lysis buffer is an isotonic ammonium chloride solution used to remove red blood cells from leukocyte preparations such as mouse spleen, bone marrow and buffy coat. The standard composition is 150 mM ammonium chloride (8.02 g/L NH4Cl), 10 mM potassium bicarbonate (1.0 g/L KHCO3) and 0.1 mM disodium EDTA, adjusted to pH 7.2-7.4. Red cells lyse osmotically within one to five minutes at room temperature because their band 3 anion exchanger and high carbonic anhydrase activity drive ammonium chloride and water into the cell, while leukocytes, which lack that transport capacity, survive.
• PBS vs DPBS: What Is Different, and Which One to UsePBS and DPBS are both phosphate-buffered saline solutions; the difference is the recipe, not the function. Dulbecco's formulation (DPBS) adds potassium chloride and carries roughly twice the phosphate of a typical PBS (about 9.5 mM versus about 4 mM), and it is sold in two versions - with calcium and magnesium, and without. In practice the with-or-without-divalent-cations choice matters far more than the PBS-or-DPBS label: use a calcium- and magnesium-free solution to wash cells before trypsinisation or EDTA dissociation, because Ca2+ and Mg2+ support the cadherin and integrin bonds you are about to break, and use the version containing calcium and magnesium when cells must stay attached and intact through the wash.