alamarblue Technical Datasheet Bio-Rad Laboratories Endeavour House, Langford Lane, Kidlington, Oxford, OXON OX5 1GE, UK antibody_sales_uk@bio-radcom Tel: +44 () 1865 8527 Copyright Bio-Rad Laboratories, Inc 216 All Rights Reserved wwwbio-rad-antibodiescom Issue No 6 Page 1 of 11
alamarblue Volume / Quantity: Product Form: BUF12A 25 ml BUF12B 1 ml Liquid Preservatives / Stabilizers: None present Product Description: alamarblue is an indicator dye which incorporates an oxidation-reduction (REDOX) indicator that both fluoresces and changes colour in response to the chemical reduction of growth medium, resulting from cell growth The alamarblue assay is designed to quantitatively measure the proliferation of various human and animal cell lines, bacteria and fungi Indications for Use: The bioassay can be used to establish proliferation or relative cytotoxicity Baseline data for predicting the toxicity of related novel agents can be compared to baseline data with known in-vivo toxicity alamarblue is for use between ph68 and ph74 Product Principle: Growing cells cause a chemical reduction of alamarblue Continued growth maintains a reduced environment (fluorescent, red) Inhibition of growth maintains an oxidized environment (non-fluorescent, blue) Data may be collected using either fluorescence-based or absorbance-based instrumentation Fluorescence is monitored at 53-56nm excitation wavelength and 59nm emission wavelength Absorbance is monitored at 57nm and 6nm Shelf life: This product is guaranteed for 12 months from date of dispatch Storage and Stability: This product may be stored for 12 months at room temperature Store at 2-8 C to increase shelf life to 2 months This product is light sensitive and should be stored in the dark Health and Safety information: The REDOX indicator in alamarblue has no current or past indication of carcinogenic capacity (A full Health and Safety assessment is available upon request) Manufactured for Bio-Rad by Trek Diagnostic System US patent 5,51,959 Please visit wwwbio-rad-antibodiescom/alamarblue for more: Calculate results online References Example calculations Frequently asked questions Issue No 6 Page 2 of 11
QC test for alamarblue Materials and Equipment: 1M potassium phosphate buffer, ph74 1ml test tube Pipette capable of accurately dispensing 4ml Plate reader with the filters 57nm and 6nm (Alternative filters can be used see bottom of page) Dynatech flat bottom plate Procedure: 1 Shake alamarblue to mix before use 2 Pipette 4ml of alamarblue into a test tube 3 Dilute to 1ml with phosphate buffer 4 Mix well 5 Pipette 1μl into each well of a clear, flat bottom microplate 6 Read absorbance at appropriate wavelengths Wavelength (nm) Average Absorbance (Standard Deviation) 54 145 (2) 57 225 (3) 6 313 (4) 63 116 (2) Expected results for Quality Control (QC) test using Dynatech flat bottomed plates NB Absorbance values may be affected by the type of plate (whether round or flat bottom) and the plate manufacturer (See Appendix 4 for further information about the effect of plates on absorbance) No QC protocol is recommended for fluorescence since fluorescence units are arbitrary and the scale used varies widely from one instrument to another For an indication of the absorbance and fluorescence values for the fully oxidized and reduced forms of alamarblue with different media, please visit the Frequently Asked Questions section found at: wwwbio-rad-antibodiescom/alamarblue Alternative wavelengths Although it is preferable to use the recommended wavelengths, a correction factor can be calculated allowing the use of one alternative filter (page 8) Recommended Alternative 57 54 6 63 57nm and 6nm are the recommended wavelengths; only one wavelength may be altered for its respective alternative wavelength Issue No 6 Page 3 of 11
Method and calculations for alamarblue Page Method for determining optimum length of incubation and plating density 4 Method for measuring cytotoxicity or proliferation using alamarblue 5 Measuring cytotoxicity or proliferation using alamarblue by Spectrophotometry 6 Equation to measure cytotoxicity 6 Equation to measure percentage reduction of alamarblue 6 Measuring cytotoxicity or proliferation using alamarblue by Fluorescence 7 Equation to measure cytotoxicity 7 Equation to measure percentage reduction of alamarblue 7 Method to measure LD5 using alamarblue 7 Measuring proliferation using spectrophotometry with different filters 8 Equation to measure cytotoxicity 8 Equation to measure percentage reduction of alamarblue 8 Method for determining optimum length of incubation and plating density The two variables which most affect the response of cells to alamarblue are length of incubation time and number of cells plated It is recommended that the plating density and incubation time be determined for each cell line using the following procedure: 1 Harvest cells which are in log phase growth stage and determine cell count Plate cells at various densities, above and below the cell density expected to be used 2 Aseptically add alamarblue in an amount equal to 1% of the volume in the well 3 Return plates to incubator Remove the plate and measure fluorescence/absorbance each hour following plating for the first 6-8 hours It is also recommended that the plate remain in incubation overnight and measurements be made the following day at 24 hours Two kinds of information can be obtained from this data: (i) (ii) for any given incubation time selected, the range in cell density relating cell number to alamarblue reduction can be determined by a linear response; for any given cell density selected, the incubation time can be determined as the time taken for the control cells to turn the indicator from the oxidized (blue) form to the fully reduced (red) form 4 Measure absorbance at a wavelength of 57nm and 6nm; or measure fluorescence with an excitation wavelength at 53-56nm and emission wavelength at 59nm 5 Calculate the percentage reduction of alamarblue at each cell density or incubation period a Equation 1 calculates percentage reduction of alamarblue using absorbance Percentage reduction of alamarblue (equation 1) = (O2 x A1) - (O1 x A2) x 1 (R1 x N2) - (R2 x N1) Where: O1 = molar extinction coefficient (E) of oxidized alamarblue (Blue) at 57nm* O2= E of oxidized alamarblue at 6nm* R1 = E of reduced alamarblue (Red) at 57nm R2= E of reduced alamarblue at 6nm A1 = absorbance of test wells at 57nm A2 = absorbance of test wells at 6nm N1 = absorbance of negative control well (media plus alamarblue but no cells) at 57nm Issue No 6 Page 4 of 11
N2 = absorbance of negative control well (media plus alamarblue but no cells) at 6nm * Only one appropriate substitute wavelength may be used Wavelength Reduced (R) Oxidized (O) 54nm 14395 47619 57nm 155677 8586 6nm 14652 117216 63nm 5494 34798 Molar extinction coefficients for alamarblue at different wavelengths b) To calculate percentage reduction of alamarblue using fluorescence, use equation 2 Percentage reduction of alamarblue (equation 2) = FI 59 of test agent Fl 59 untreated control x 1 FI 59 of 1% reduced alamarblue TM Fl 59 untreated control Where: FI 59 = Fluorescent Intensity at 59nm emission (56nm excitation) 6 Plot a graph of percentage reduction of alamarblue at each cell density or incubation period a) For experiments to determine optimum cell density, plot log of cell density on the x axis, and percentage reduction on the y axis; b) For experiments to determine the optimum incubation time, plot the number of hours on the x axis, and the percentage reduction on the y axis c) Use the graphs to determine optimum cell density or incubation period (Example data is given in appendix 1) General method for measuring cytotoxicity or proliferation using alamarblue 1 Harvest cells which are in the log phase of growth and determine cell count Adjust the cell count to 1x1 4 (suggested cell density) The optimum cell density may vary between cell types 2 Plate cells and expose to test agent as determined by researcher For determining the effect of a test agent on cell growth, ensure correct controls are included eg stimulated vs unstimulated cells 3 Mix by shaking, then aseptically add alamarblue in an amount equal to 1% of the volume in the well 4 Incubate cultures with alamarblue for 4-8 hours NB The optimum incubation time may vary between cell types 5 Measure cytotoxicity or proliferation using spectrophotometry of fluorescence Issue No 6 Page 5 of 11
Method for measuring cytotoxicity or proliferation using alamarblue by spectrophotometry 6 Measure absorbance at wavelengths of 57nm and 6nm after required incubation Use a blank of media only To calculate the percent difference in reduction between treated and control cells in cytotoxicity and proliferation assays (Equation 3): Percentage difference between treated and control cells (equation 3) = (O2 x A1) - (O1 x A2) x 1 (O2 x P1) - (O1 x P2) Where: O1 = molar extinction coefficient (E) of oxidized alamarblue (Blue) at 57nm* O2= E of oxidized alamarblue at 6nm* A1 = absorbance of test wells at 57nm A2 = absorbance of test wells at 6nm P1 = absorbance of positive growth control well (cells plus alamarblue but no test agent) at 57nm P2 = absorbance of positive growth control well (cells plus alamarblue but no test agent at 6nm * Only one appropriate substitute wavelength may be used Wavelength Reduced (R) Oxidized (O) 54nm 14395 47619 57nm 155677 8586 6nm 14652 117216 63nm 5494 34798 Molar extinction coefficients for alamarblue at different wavelengths Alternatively, it may be useful to calculate the percent reduction of alamarblue (Equation 1): Percentage reduction of alamarblue (equation 1) = (O2 x A1) - (O1 x A2) x 1 (R1 x N2) - (R2 x N1) Where: O1 = molar extinction coefficient (E) of oxidized alamarblue (Blue) at 57nm* O2= E of oxidized alamarblue at 6nm* R1 = E of reduced alamarblue (Red) at 57nm R2= E of reduced alamarblue at 6nm A1 = absorbance of test wells at 57nm A2 = absorbance of test wells at 6nm N1 = absorbance of negative control well (media plus alamarblue but no cells) at 57nm N2 = absorbance of negative control well (media plus alamarblue but no cells) at 6nm * Only one appropriate substitute wavelength may be used For an Excel calculator and example calculations, please visit wwwbio-rad-antibodiescom/alamarblue Issue No 6 Page 6 of 11
General method for measuring cytotoxicity or proliferation using alamarblue by fluorescence 6 Read fluorescence at excitation 56nm, emission 59nm To calculate percent difference in reduction between treated and control cells in cytotoxicity/ proliferation assays use the following formula (Equation 4): Percentage difference between treated and control cells (equation 4) = FI 59 of test agent x 1 FI 59 of untreated control Where: FI 59 = Fluorescent Intensity at 59nm emission (56nm excitation) If required, equation 2 can be used to find the percentage reduction of alamarblue using fluorescence: Percentage reduction of alamarblue (equation 2) = FI 59 of test agent Fl 59 untreated control x 1 FI 59 of 1% reduced alamarblue TM Fl 59 untreated control Where: FI 59 = Fluorescent Intensity at 59nm emission (56nm excitation) For equation 2, it is necessary to include the fluorescence value for alamarblue in its fully reduced form To produce the 1% reduced form of alamarblue, simply autoclave a sample containing media and alamarblue for 15 minutes Since fluorescence units are arbitrary and may therefore vary depending upon instrument set up, it is important that users determine the fluorescence reading for 1% reduction using the same media and instrument as for their samples Worked calculations and Frequently Asked Question at wwwbio-rad-antibodiescom/alamarblue Method to determine LD5 using alamarblue Use semi-log graph paper to plot the percent of untreated control (using equation 5) for each dilution of a given test agent on the y-axis vs the concentration of the test agent on the x-axis To determine the LD5 endpoint from the graph, read from where the 5 percent point intercepts the Dose Response Curve to the concentration along the x-axis This concentration is the LD5 value 1 % Growth Control 75 5 25-1 -9-8 -7-6 Log [Drug] (M) Determination of Doxorubicin LD5 using alamarblue Issue No 6 Page 7 of 11
Calculations for using alamarblue in Spectrophotometry with different filters 1 Make up alamarblue (AB) as directed in the package insert (1/1 dilution in 1μI media) 2 Measure the absorbance of alamarblue in media at the lower wavelength (LW) filter and at the higher wavelength (HW) filter 3 Measure the absorbance of 1ul media only (blank) at the two wavelengths 4 Subtract the absorbance values of media only from the absorbance values of alamarblue in media AO LW = Absorbance of AB in media Absorbance of media only AO HW = Absorbance of AB in media Absorbance of media only Where: AO LW = absorbance of oxidized form at the lower wavelength AO HW = absorbance of oxidized form at the higher wavelength 5 Calculate correction factor (R ): Correction factor for different filters: R = AO LW /AO HW 6 Calculate percentage difference in reduction between treated and control cells in cytotoxicity/proliferation assays: Percentage difference in reduction = A LW (A HW x R ) for test well x 1 (Equation 5) A LW (A HW x R ) for control well Where: A LW = absorbance at lower wavelength minus the media blank A HW = absorbance at higher wavelength minus the media blank R = Correction factor (calculated in step 5) 7 If the alternative calculation to find the percent reduction of alamarblue is required, use the following equation: Percentage reduction of alamarblue = [ A LW - (A HW x R ) ] x 1 (Equation 6) alamarblue Appendix Appendix 1 Appendix 2 Appendix 3 Appendix 4 Worked example to determine optimum cell density and incubation time for A549 cells, using alamarblue and spectrophotometry Worked example to determine optimum cell density and incubation time for P388 cells, using alamarblue and spectrophotometry Example alamarblue reduction curves for four cell lines using fluorescence Effect of storage plates on spectrophotometric readings Page 9 1 11 11 Issue No 6 Page 8 of 11
Appendix 1: Worked example to determine optimum cell density and incubation time for A549 cells, using alamarblue and spectrophotometry The cell line A549 is a monolayer culture The method given on page 4 was followed to find the percentage reduction of alamarblue after incubation with A549 cells at different densities and for different incubation periods Absorbance values at 57nm and 6nm after blanking with media only for A549 cells Time (hours) Blue In Media Absorbance values at 57nm 5 Blue In Media Absorbance values at 6nm 5 336 336 338 348 372 441 439 443 459 496 2 334 339 352 432 54 44 425 421 349 267 4 333 346 365 489 59 432 411 397 265 162 55 321 344 366 511 573 424 397 377 211 135 2 322 438 51 518 486 412 271 18 12 112 Using absorbance data from the sample data set, percent reduction of alamarblue was calculated using equation 1: Percentage reduction of alamarblue after incubation with A549 cells at different cell densities and incubation periods Percentage reduction of alamarblue Time (hours) 5 These values are plotted to produce Figures 1a & 1b 63 61 6 57 2 86 115 354 657 4 119 173 577 899 55 136 24 7 918 2 496 762 883 87 Figure 1a Figure 1b % Reduced 1 9 8 7 6 5 4 3 2 1 2 4 55 2 5 % Reduction 1 9 8 7 6 5 4 3 2 1 5 hours 2 hours 4 hours 55 hours 2 hours Hours Cell Density () Graphs showing percentage of alamarblue reduction for A549 cells with different incubation periods (Figure 1a) and at different initial cell densities (Figure 1b) Please refer to the Worked Example section of wwwbio-rad-antibodiescom/alamarblue for notes to accompany these graphs Issue No 6 Page 9 of 11
Appendix 2: Worked example to determine optimum cell density and incubation time for P388 cells, using alamarblue and spectrophotometry The cell line P388 is a suspension cell line The method given on page 4 was followed to find the percentage reduction of alamarblue after incubation with P388 cells at different densities and for different incubation periods Absorbance values at 57nm and 6nm after blanking with media only for P388 cells Time (hours) Absorbance values at 57nm Absorbance values at 6nm Blue In Media 5 Blue In Media 5 336 342 334 332 328 441 451 438 433 422 2 334 34 333 335 335 44 448 435 422 44 4 333 339 332 339 346 432 444 432 414 391 55 321 331 325 335 344 424 435 424 44 377 2 322 332 328 381 434 412 423 415 337 253 Using absorbance data from the sample data set, percent reduction of alamarblue was calculated using equation 1: Percentage reduction of alamarblue for P388 cells at a range of cell densities following different incubation periods Time (hours) Percentage reduction of alamarblue 5 59 6 63 7 2 59 63 83 16 4 63 66 12 145 55 61 64 19 162 2 81 84 295 513 These values are plotted to produce Figures 2a & 2b Figure 2a Figure 2b 6 6 5 5 % Reduced 4 3 2 5 % Reduced 4 3 2 Hours 2 Hours 4 Hours 55 Hours 2 Hours 1 1 2 4 55 2 5 Hours Cell Density () Graphs showing percentage of alamarblue reduction for P388 cells with different incubation periods (Figure 2a) and at different initial cell densities (Figure 2b) Please refer to the Worked Example section of wwwbio-rad-antibodiescom/alamarblue for notes to accompany these graphs Issue No 6 Date:23rd May 2164 Page 1 of 11
Appendix 3: Example alamarblue reduction curves for four cell lines using fluorescence Examples of reduction curves are included to demonstrate the usefulness of the alamarblue assay for measuring cell proliferation Detection of cell growth of four cell lines using alamarblue 6 Fluorescence 4 3 2 HT29 HT18 DU145 a549 1 156 312 625 125 25 Initial Cell Density Appendix 4: Effect of storage plates on spectrophotometric readings The absorbance readings of alamarblue may be affected by the type of plates which are used for experiments A series of absorbance readings were taken for the oxidized and reduced forms of alamarblue using a range of different types of plates Following the readings on day 1, plates were covered in foil and refrigerated prior to further readings on days 2 & 3 See Frequently Asked Question 11 at wwwbio-rad-antibodiescom/alamarblue for more information about storing plates prior to collecting data Plate Type Dynatech Flat Bottom Corning Flat Bottom Corning Round Bottom Day Absorbance BLUE (Oxidized) RED (Reduced) 54nm 57nm 6nm 63nm 54nm 57nm 6nm 63nm Day 1 (3) (5) (7) (3) (2) (27) (2) () 298 496 78 236 693 117 126 75 Day 2 (3) (4) (6) (2) (2) (27) (8) (9) 294 484 692 227 697 118 164 118 Day 3 (3) (6) (8) (3) (1) (24) (8) (9) 296 486 691 231 734 138 199 149 Day 1 (2) (4) (5) (2) (2) (24) (3) (4) 21 335 474 169 53 772 137 15 Day 2 (2) (3) (4) (1) (2) (27) (4) (4) 21 329 458 161 58 822 193 159 Day 3 (2) (3) (5) (2) (2) (35) (4) (3) 2 322 444 16 6 823 21 172 Day 1 (1) (2) (2) (1) (14) (18) (2) () 38 635 913 3 87 1266 151 84 Day 2 (2) (3) (4) (2) (11) (14) (2) (2) 39 641 914 295 85 1241 146 83 Day 3 (4) (6) (8) (4) (17) (21) (7) (6) 39 646 916 32 86 1237 159 94 Absorbance of alamarblue oxidized and reduced forms for a range of plates, including those refrigerated prior to data collection 1ul of RPMI 164 with MOPS ph7, no phenol red Standard deviations are in parentheses (calculated for n=8) Issue No 6 Page 11 of 11