TITLE: CODEX Preparation of Reporter 96-Well Plates
AUTHORS: Jerelyn Nick, Marda Jorgensen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">A CODEX® Reporter is a fluorescent dye conjugated to an oligonucleotide that is complementary to a specific oligonucleotide barcoded antibody. This protocol describes how to prepare 96-well plates of Reporter Mix for use in a CODEX® run. Each well contains Reporters are grouped together in mixtures of three per cycle plus the nuclear stain. During each cycle the Codex instrument dispenses one well on the tissue sample and images the 3 markers and the nuclear stain. At the end of the cycle the Reporters are removed from the tissue by a gentle wash.</div></div>

[STEPS]
?. Prepare the Reporter Stock Solution (Based on the number of cycles and coverslips to be run ):   ABCDEFGHIJ1  Prepare Reporter Stock Solution  2Number of cycles (wells)  5    9    10    15    18    20    27    36    45  3     cycles (wells)4  Nuclease Free Water    1220    2196    2440    3660    4392    4880    6588    8784    10980  5  10X CODEX buffer    150    270    300    450    540    600    810    1080    1350  6  Assay Reagent    125    225    250    375    450    500    675    900    1125  7  Nuclear Stain    5    9    10    15    18    20    27    36    45  8  Total    1500    2700    3000    4500    5400    6000    8100    10800    13500  9Example: # Coverslips@ 9 cycles     1 coverslip          2 coverslips     3 coverslips4 coverslips5 coverslips
ABCDEFGHIJ1  Prepare Reporter Stock Solution  2Number of cycles (wells)  5    9    10    15    18    20    27    36    45  3     cycles (wells)4  Nuclease Free Water    1220    2196    2440    3660    4392    4880    6588    8784    10980  5  10X CODEX buffer    150    270    300    450    540    600    810    1080    1350  6  Assay Reagent    125    225    250    375    450    500    675    900    1125  7  Nuclear Stain    5    9    10    15    18    20    27    36    45  8  Total    1500    2700    3000    4500    5400    6000    8100    10800    13500  9Example: # Coverslips@ 9 cycles     1 coverslip          2 coverslips     3 coverslips4 coverslips5 coverslips
?. Prepare the Reporter Master Mix Aliquots for Each Cycle
?. Label an amber microcentrifuge tube for each cycle.  Include tubes for the blank cycles at the beginning and end of the experiment.
?. Aliquot  Reporter Stock Solution into each tube following the chart below (volumes will vary depending on the number of reporters used in an individual cycle (see CODEX Cycle Template).  ABCDE1Reporter Solutions: 
Label a 1.5 ml amber tube for each cycle23 Reporters2 Reporters  1&nbsp; Reporter  (Blank) 0 Reporters31 Plate  
(will add 5ul each reporter)  235 ul    240    245    250  42 Plates
(will add 10 ul each reporter)  470 ul    480    490    500  53 Plates 
(will add 15 ul each reporter)  705 ul    720    735    750  64 Plates
 (will add 20 ul each reporter)  940 ul    960    980    1000  75 Plates 
(will add 25 ul each reporter)  1175ul    1200    1225    1250
ABCDE1Reporter Solutions: 
Label a 1.5 ml amber tube for each cycle23 Reporters2 Reporters  1&nbsp; Reporter  (Blank) 0 Reporters31 Plate  
(will add 5ul each reporter)  235 ul    240    245    250  42 Plates
(will add 10 ul each reporter)  470 ul    480    490    500  53 Plates 
(will add 15 ul each reporter)  705 ul    720    735    750  64 Plates
 (will add 20 ul each reporter)  940 ul    960    980    1000  75 Plates 
(will add 25 ul each reporter)  1175ul    1200    1225    1250
?. Add appropriate Reporters to the Reporter Stock Solution aliquots to make a Master Mix for each cycle
?. Place reporters on ice.
?. Briefly, Spin tubes using a bench top centrifuge. Return Reporters to ice.
?. Pipette Akoya Reporters into the labeled amber tubes containing Reporter Stock Solution. Use volumes listed in step 2.2. column A based on number of reporters in a particular cycle.Follow the cycle template or the reporter tray template (below).  ABCDE1  CODEX Cycle Template  2  Nuclear
  Stain    AF
  750    550    Cy5  3cycle  1      Blank cyclein Reporter StockNo ReporterNo reporterNo Reporter4cycle 2   (example: 3 antibodies,-750, 550, and Cy5)in Reporter StockReporter 1   (matches antibody1)Reporter 2   ( matches antibody2)Reporter 3   ( matches antibody3)5cycle 3 (example 2 antibodies- 550 and Cy5)in Reporter StockNo Antibody in this wavelengthReporter 4   ( matches antibody4)Reporter 5   ( matches antibody5)6cycle 4 (example 1 antibody- 550)in Reporter StockNo Antibody in this wavelengthReporter 6 ( matches antibody6)No Antibody in this wavelength75                    8  6                      9  7                      10  8                      11  *                      12last cycle     Blank cyclein Reporter StockNo ReporterNo reporterNo Reporter  ABCDEFGH1Reporter  Tray
 Template2Cycle 1&nbsp;Cycle 2&nbsp;Cycle 3&nbsp;Cycle 4Cycle 5+Cycle *Cycle*+1dye/fluor3BlankAntibody 1 (Reporter 1)Antibody 4
(Reporter 4)label any EMPTY
  channels&nbsp;  Blank&nbsp;Cy74 Antibody 2 
(Reporter 2)Antibody 5
 (Reporter 5)Antibody 7
(Reporter 7)   5505 Antibody 3 (Reporter 3)Antibody 6
(Reporter 6)...etc   Cy5&nbsp;
ABCDE1  CODEX Cycle Template  2  Nuclear
  Stain    AF
  750    550    Cy5  3cycle  1      Blank cyclein Reporter StockNo ReporterNo reporterNo Reporter4cycle 2   (example: 3 antibodies,-750, 550, and Cy5)in Reporter StockReporter 1   (matches antibody1)Reporter 2   ( matches antibody2)Reporter 3   ( matches antibody3)5cycle 3 (example 2 antibodies- 550 and Cy5)in Reporter StockNo Antibody in this wavelengthReporter 4   ( matches antibody4)Reporter 5   ( matches antibody5)6cycle 4 (example 1 antibody- 550)in Reporter StockNo Antibody in this wavelengthReporter 6 ( matches antibody6)No Antibody in this wavelength75                    8  6                      9  7                      10  8                      11  *                      12last cycle     Blank cyclein Reporter StockNo ReporterNo reporterNo Reporter
ABCDEFGH1Reporter  Tray
 Template2Cycle 1&nbsp;Cycle 2&nbsp;Cycle 3&nbsp;Cycle 4Cycle 5+Cycle *Cycle*+1dye/fluor3BlankAntibody 1 (Reporter 1)Antibody 4
(Reporter 4)label any EMPTY
  channels&nbsp;  Blank&nbsp;Cy74 Antibody 2 
(Reporter 2)Antibody 5
 (Reporter 5)Antibody 7
(Reporter 7)   5505 Antibody 3 (Reporter 3)Antibody 6
(Reporter 6)...etc   Cy5&nbsp;
?. Prepare the 96-well Reporter Plate:Pipette 245 ul of Reporter containing Master Mix into the corresponding cycle well .
?. Use a different 96-well plate for each Reporter plate prepared. Lightly cover entire rows of wells (i.e. B1-B12) with strips of foil seal.Keep the filled 96-well plates IN THE DARK to avoid photo bleaching.Store at 4˚C for up to two weeks.