RPMI 1640 Medium with Phenol Red and L-Glutamine

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• RPMI 1640 MediumRPMI 1640 is a basal cell culture medium developed at Roswell Park Memorial Institute in 1966 for the culture of human leukocytes in suspension. It contains 2,000 mg/L glucose (11.1 mM), 2,000 mg/L sodium bicarbonate (23.8 mM) buffered for a 5% CO2 atmosphere, unusually high phosphate (about 5.6 mM), low calcium (about 0.42 mM), and a distinctive component set that includes reduced glutathione, biotin, vitamin B12, para-aminobenzoic acid and hydroxyproline. It is the standard medium for lymphocytes, hybridomas, and most suspension-adapted haematopoietic and lymphoid cell lines, normally supplemented with 10% fetal bovine serum.
• Phenol Red in Cell Culture MediaPhenol red (phenolsulfonphthalein) is a pH indicator added to cell culture media at roughly 5-15 mg/L, where it turns yellow below about pH 6.8 and pink to fuchsia above about pH 8.2, with the normal red-orange of healthy medium sitting near pH 7.4. Medium turning yellow means acidification - almost always lactate from cell metabolism, from an overgrown culture or from bacterial contamination - while medium turning pink or purple means the medium has gone alkaline, nearly always because CO2 has escaped from the bicarbonate buffer. Phenol red does no buffering itself and is purely diagnostic; use phenol-red-free medium for fluorescence and absorbance assays, and for oestrogen-responsive cell work, where the dye interferes with the readout.
• 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.
• Essential vs Non-Essential Amino Acids in Cell CultureIn cell culture, an amino acid is called essential if cultured mammalian cells cannot make enough of it and it must be supplied in the medium - thirteen of them, the set Harry Eagle defined, including arginine, cystine, tyrosine and glutamine that are not classed as dietary essentials. The seven non-essential amino acids - glycine, alanine, asparagine, aspartic acid, glutamic acid, proline and serine - can be synthesised by most cells, and are supplied anyway because making them consumes carbon, ATP and reducing equivalents that would otherwise go into growth. A MEM NEAA 100X supplement contains all seven at 10 mM, giving 0.1 mM of each at working strength; add it to MEM or DMEM, which contain few or none, and leave it out of Ham's F-12, IMDM and DMEM/F-12, which already carry all seven.