RPMI-1640 without L-Glutamine and Sodium Bicarbonate (Powder)

Product#: HM-AT028A
$9.49
HM-AT028A
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RPMI-1640
Without L-Glutamine and Sodium bicarbonate

Product Code: HM-AT028A

Product Description:


Roswell Park Memorial Institute (RPMI) media are a series of media developed by Moore et al for the culture of human normal and neoplastic cells in vitro. RPMI 1640 is the most commonly used medium in the series. A modification of McCoy's 5A medium, the medium was specifically designed to support the growth of human lymphoblastoid cells in suspension culture. Presently the medium is extensively used for a wide range of anchorage dependant cell lines. The medium needs to be supplemented with 5-20% fetal bovine serum. The medium is also known to support growth of cells in the absence of serum.

AT028A is RPMI 1640 without L-glutamine. Users are advised to review the literature for recommendations regarding medium supplementation and physiological growth requirements specific for different cell lines.

Composition:

Ingredients mg/L   Ingredients mg/L
INORGANIC SALTS   VITAMINS  
Calcium nitrate tetrahydrate  100.000   Choline chloride 3.000
Magnesium sulphate anhydrous  48.840   D-Biotin 0.200
Potassium chloride  400.000   D-Pantothenic acid 0.250
Sodium chloride  6000.000   Folic acid  1.000
Sodium dihydrogen phosphate anhydrous  800.000   Niacinamide  1.000
AMINO ACIDS     Pyridoxine hydrochloride  1.000
Glycine  10.000   Riboflavin  0.200
L-Arginine hydrochloride  241.000   Thiamine hydrochloride  1.000
L-Asparagine  50.000   Vitamin B12  0.005
L-Aspartic acid  20.000   i-Inositol  35.000
L-Cystine dihydrochloride  65.200   p-Amino benzoic acid (PABA)  1.000
L-Glutamic acid  20.000   OTHERS  
L-Histidine hydrochloride monohydrate  20.960   D-Glucose  2000.000
L-Hydroxyproline  20.000   Glutathione reduced  1.000
L-Isoleucine  50.000   Phenol red sodium salt  5.300
L-Leucine  50.000      
L-Lysine hydrochloride  40.000      
L-Methionine  15.000      
L-Phenylalanine  15.000      
L-Proline  20.000      
L-Serine  30.000      
L-Threonine  20.000      
L-Tryptophan  5.000      
L-Tyrosine sodium salt  28.830      
L-Valine  20.000      


Directions:
  1. Suspend 10.1gms in 900ml tissue culture grade water with constant, gentle stirring until the powder is completely dissolved. Do not heat the water.
  2. It may be necessary to lower the pH to 4.0 with 1N HCl to completely dissolve this product. After it has dissolved completely, the pH can be raised to 7.2 with 1N NaOH prior to the addition of sodium bicarbonate
  3. Add 2.0gms of sodium bicarbonate powder or 26.7ml of 7.5% Sodium bicarbonate solution (TCL013) and 10.3ml of 200mM L-glutamine solution (TCL012) for 1 litre of medium and stir until dissolved.
  4. Adjust the pH to 0.2-0.3 pH units below the desired pH using 1N HCl or 1N NaOH since the pH tends to rise during filtration.
  5. Make up the final volume to 1000ml with tissue culture grade water.
  6. Sterilize the medium immediately by filtering through a sterile membrane filter with a porosity of 0.22 micron or less, using positive pressure rather than vacuum to minimize the loss of carbon dioxide.
  7. Aseptically add sterile supplements as required and dispense the desired amount of sterile medium into sterile containers.
  8. Store liquid medium at 2-8°C and in dark till use.

Material required but not provided:
  • Tissue culture grade water (TCL010)
  • Sodium bicarbonate (TC230)
  • Sodium bicarbonate solution, 7.5% (TCL013)
  • L-Glutamine Powder (TC243)
  • L-Glutamine solution 200mM (TCL012)
  • 1N Hydrochloric acid (TCL003)
  • 1N Sodium hydroxide (TCL002)
  • Foetal bovine serum (RM1112/RM10432)

Quality Control:
  • Appearance: Off-white to Creamish white, homogenous powder.
  • Solubility: Clear solution at 10.1 gms/L.
  • pH without Sodium Bicarbonate: 7.20 -7.80
  • pH with Sodium Bicarbonate: 7.40 -8.00
  • Osmolality without Sodium Bicarbonate: 230.00 -270.00
  • Osmolality with Sodium Bicarbonate: 280.00 -320.00
  • Cultural Response: The growth promotion capacity of the medium is assessed qualitatively by analyzing the cells for the morphology and quantitatively by estimating the cell counts and comparing it with a control medium through minimum three subcultures.
  • Endotoxin Content: NMT 5EU/ml

Storage and Shelf Life:
  1. All the powdered media and prepared liquid culture media should be stored at 2-8°C. Use before the expiry date. Inspite of above recommended storage condition, certain powdered medium may show some signs of deterioration /degradation in certain instances. This can be indicated by change in colour, change in appearance and presence of particulate matter and haziness after dissolution.
  2. Preparation of concentrated medium is not recommended since free base amino acids and salt complexes having low solubility may precipitate in concentrated medium.
  3. pH and sodium bicarbonate concentration of the prepared medium are critical factors affecting cell growth. This is also influenced by amount of medium and volume of culture vessel used (surface to volume ratio). For example, in large bottles, such as Roux bottles pH tends to rise perceptibly as significant volume of carbon dioxide is released. Therefore, optimal conditions of pH, sodium bicarbonate concentration, surface to volume ratio must be determined for each cell type. We recommend stringent monitoring of pH. If needed, pH can be adjusted by using sterile 1N HCl or 1N NaOH or by bubbling in carbon dioxide.
  4. If required, supplements can be added to the medium prior to or after filter sterilization observing sterility precautions. Shelf life of the medium will depend on the nature of supplement added to the medium.
 

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