Make double-stranded salmon sperm DNA 1.
{"action": "make", "output": "", "reagent": ["double-stranded", "salmon sperm DNA 1"], "length": [""]}
Add 100 μL/well of 100 μg/mL salmon sperm DNA to a 96-well Microtest assay plate.
{"action": "add", "output": "", "volume": ["\u03bcL"], "container": ["96-well"]}
Wrap the plate with plastic wrap .
{"action": "wrap", "output": "", "length": [""], "reagent": [""]}
incubate at 4 °C overnight #####%%%%% output "the coating antibody solution" are used in line10
{"action": "incubate", "output": "the coating antibody solution", "temperature": ["4 \u00b0C"]}
Discard the coating antibody solution . #####%%%%% output "1x PBS-Tween" are used in line12
{"action": "remove", "output": "1x PBS-Tween", "reagent": ["the coating antibody solution"]}
wash the plate with 1x PBS-Tween 6 times
{"action": "wash", "output": "", "reagent": ["1x PBS-Tween"]}
Dry the plate .
{"action": "rinse", "output": "", "container": ["the plate"]}
add 100 μL of blocking solution per well to the plate
{"action": "add", "output": "", "container": ["well"]}
Incubate the plate at room temperature (RT) for 1.5 h. 6. #####%%%%% output "the blocking solution" are used in line20
{"action": "incubate", "output": "the blocking solution", "container": ["the plate"], "temperature": ["room temperature (RT)"], "time": [""]}
Discard the blocking solution . #####%%%%% output "1x PBS-Tween" are used in line22
{"action": "remove", "output": "1x PBS-Tween", "reagent": ["the blocking solution"]}
wash the plate with 1x PBS-Tween 5 times
{"action": "wash", "output": "", "reagent": ["1x PBS-Tween"]}
Dry the plate .
{"action": "rinse", "output": "", "container": ["the plate"]}
keep it at 4 °C for later use #####%%%%% output "the spleen" are used in line28
{"action": "keep", "output": "the spleen", "temperature": ["4 \u00b0C"]}
Harvest the spleen . #####%%%%% output "spleen pieces" are used in line30
{"action": "export", "output": "spleen pieces", "reagent": ["the spleen", "the spleen"], "time": [""]}
create a single-cell suspension by gently smashing spleen pieces with the frosted surface of a pair of microscope slides in 5 mL of DMEM #####%%%%% output "a single-cell suspension" are used in line44
{"action": "create", "output": "a single-cell suspension", "reagent": ["spleen pieces"]}
Transfer the cells into 50 mL conical tubes .
{"action": "transfer", "output": "", "container": ["the cells"]}
spin down the cells at 300 RCF for 5 min at 4 °C
{"action": "turn", "output": "", "temperature": ["4 \u00b0"]}
Discard the supernatant with aspiration without disturbing the pellet.

Re-suspend the cells with 5 mL of 0.17 M ammonium chloride
{"action": "suspend", "output": "", "container": ["the cells"], "concentration": ["0.17 M ammonium chloride"]}
keep the cells on ice for 5 min.
{"action": "keep", "output": "", "time": ["5 min"]}
Add 15 mL DMEM to the cells .

Discard the supernatant . #####%%%%% output "the supernatant" are used in line46
{"action": "remove", "output": "the supernatant", "reagent": ["a single-cell suspension"]}
suspend the cells with 20 mL of DMEM
{"action": "suspend", "output": "", "reagent": ["DMEM", "the supernatant"]}
count the cells #####%%%%% output "the cells" are used in line64
{"action": "estimate", "output": "the cells", "reagent": [""]}
Re-suspend 2 x 10^7 cells in 2 mL of 10% DMEM .
{"action": "replace", "output": "", "concentration": ["10%"]}
Add 50 μL/well of the serial dilutions on the DNA-coated plate .
{"action": "add", "output": "", "volume": ["50"], "container": ["DNA-coated"]}
Incubate the cells at 30 °C for 2 h in a cell-culture incubator with 6% CO2.
{"action": "incubate", "output": "", "temperature": ["30 \u00b0C"], "time": ["2 h"]}
Add 50 μL/well of biotin-conjugated anti-IgM IgG (1:350 in 10% DMEM) to the cells.
{"action": "add", "output": "", "volume": ["50"], "container": ["the cells"]}
Centrifuge the cells at 300 RCF for 5 min at 4 °C . #####%%%%% output "CO2" are used in line60
{"action": "centrifuge", "output": "CO2", "container": ["the cells"], "force": ["300 RCF"], "temperature": ["4 \u00b0C"], "reagent": [""], "time": [""]}
incubate the cells overnight in a cell-culture incubator with 6% CO2
{"action": "incubate", "output": "", "reagent": ["CO2"], "temperature": [""], "time": [""]}
Discard the cells .
{"action": "remove", "output": "", "container": ["the cells"], "volume": [""]}
wash the plates 10 times with 10x PBS-Tween 20
{"action": "wash", "output": "", "time": [""], "reagent": ["the cells"]}
Dry the plates . #####%%%%% output "streptavidin alkaline phosphatase (1:1,000 [in 1]% BSA/PBS)" are used in line68
{"action": "rinse", "output": "streptavidin alkaline phosphatase (1:1,000 [in 1]% BSA/PBS)", "container": ["the plates"]}
add 50 μL of streptavidin alkaline phosphatase (1:1,000 in 1% BSA/PBS) to the plate
{"action": "add", "output": "", "reagent": ["streptavidin alkaline phosphatase 1:1,000 [in 1]% BSA/PBS"], "container": ["the plate"]}
Incubate the plate at RT for 1 h . #####%%%%% output "10x PBS-Tween 20" are used in line72
{"action": "incubate", "output": "10x PBS-Tween 20", "container": ["the plate"], "temperature": ["RT"], "time": ["1 h"]}
wash the plate 10 times with 10x PBS-Tween 20
{"action": "wash", "output": "", "reagent": ["10x PBS-Tween 20"]}
Dry the plate . #####%%%%% output "AMP buffer" are used in line76
{"action": "rinse", "output": "AMP buffer", "container": ["the plate"]}
add 50 μL/well of 1 mg/mL BCIP in AMP buffer to develop the plate #####%%%%% output "the plate" are not used
{"action": "add", "output": "the plate", "reagent": ["AMP buffer"]}
When the spots are clearly visible under a dissecting microscope, stop the development by discarding the BCIP solution .

Spots can be counted using a dissecting microscope .
