Bio-Plex Multiplex Cytokine Assay Protocol Outline

For full instructions see BioPlex Binder or the protocol online at BioRad.com
Bring all buffers and diluents to room temp prior to use. Turn on the Bio-Plex instrument
at least 40 minutes prior to plate reading. The beginning of the detection antibody
step is a good time!
PREPARE STANDARDS
Recommend using the standard lo!" P#T setting and a standard cur$e starting at the
cyto%ine $alue gi$en on the insert for tissue culture supernatants. & standard cur$e using
the high P#T setting and starting at a '0-fold lo!er concentration of cyto%ine is
recommended for serum( or plasma samples see belo!"
Standard Curve or !se On Standard "#o$% PMT Settin&
'" Reconstitute standard by adding '(( l of media( or standard diluent to the standard
$ial.
a" )ortex gently for * sec.
b" +ncubate on ice for ,0--0 min.
," Prepare a 4-fold serial dilution as sho!n. .nless stated in %it( /tandard ' !ill be
-,(000 pg0m1.
REMEMBER) The calculated concentrations of your samples !ill only be accurate( if
the standards are diluted in the /&#2 media as the one found in your samples.
Tissue culture supernatants-use the same tissue culture media that !as utili3ed to gro!
your cells.
/erum( or plasma samples-use the /T&45&R5 5+1.24T from the species-specific
serum diluent %it. 6arm the diluent bottle at -789 if there is any precipitate.
Standard Curve or !se On *i&+ PMT Settin& "or,erly (-.-/0.(( p&1,l Standard
Ran&e%
'" Reconstitute standard by adding '(( l of media( or standard diluent to the standard
$ial.
a" )ortex gently for * sec.
b" +ncubate on ice for ,0--0 min.
," Prepare a 4-fold serial dilution as sho!n. .nless stated in %it( /tandard ' !ill be -(,00
pg0m1.
REMEMBER) The calculated concentrations of your samples !ill only be accurate( if
the standards are diluted in the /&#2 media as the one found in your samples.
Tissue culture supernatants-use the same tissue culture media that !as utili3ed to gro!
your cells.
/erum( or plasma samples-use the /T&45&R5 5+1.24T from the species-specific
serum diluent %it. 6arm the diluent bottle at -789 if there is any precipitate.
PREPARE SAMP#ES
'" Tissue 9ulture /upernatants -9yto%ine le$els in :neat; samples are sometimes abo$e
the range of the standard cur$e. /amples may therefore need to be diluted before
assaying. *0 ul sample is added to each !ell.
," /erum0Plasma - BioRad strongly recommends diluting serum0plasma samples '<4 in
the species=specific serum /&#P12 5+1.24T. The sample diluent can be !armed
at -789 to dissol$e precipitated material in the bottom of the bottle. *0 ul 5+1.T25
sample is added to each !ell.
PREPARE BEADS
'" 5etermine the total number of !ells that !ill be re>uired.
a" &s a precaution( add . extra !ells for e$ery ? !ells actually re>uired.
," 2ortex the anti-cyto%ine bead ,*x" stoc% solution at medium speed for ,0 sec.
-" Prepare a ,*-fold !or%ing dilution of the anti-cyto%ine bead ,*x" stoc% solution in
Bio-Plex Assay Bu33er A.
a" 5ocument all $olume calculations.
b" Protect the beads from light.
c" @eep all bead solutions on ice !hen not in use.
ASSA4
'" 9o$er all unneeded !ells !ith plastic adhesi$e plate sealer.
," /et the $acuum pressure to . inc+es *& using a standard flat bottom AB-!ell plate.
9hec% before each $acuum step to pre$ent damaging filter plate.
-" Pre-!et the filter plate !ith 5(( l of Bio-Plex Assay Bu33er-
4" )acuum filter. Blot the bottom of plate after this and e$ery $acuum filtration.
*" &dd Beads.
a" 2ortex the !or%ing Bead solution at medium speed for ,0 sec and add '( l to
each !ell.
B" )acuum filter.
7" Filter !ash .6 !ith 5(( l of Bio-Plex 7as+ Bu33er.
?" &dd standards.
a" 2ortex 3or ' sec and immediately add '( l to the appropriate !ells.
b" Remember to also include the appropriate blan%s.
A" &dd samples.
a" 2ortex 3or ' sec and immediately add '( l to the appropriate !ells.
'0" 9o$er the !ells !ith plastic adhesi$e plate sealer( and blot the bottom of the plate.
''" 9o$er !ith foil and incubate for /( ,in at roo, te,perature !ith sha%ing.
a" /lo!ly ramp up to 505(( rp, and sha%e at high speed for /( sec
b" Reduce speed to /(( rpm for the remainder of incubation
PREPARE DETECT8ON ANT8BOD4 "D!R8N9 PRECEED8N9 8NC!BAT8ON
AND 5( M8N PR8OR TO !SE%
'" 5etermine the total number of !ell that !ill be re>uired.
a" &s a precaution( add . extra !ells for e$ery ? !ells actually re>uired.
b" 9ently vortex the multiplex detection antibody stoc% solution.
," C4ote< the concentration of the detection antibody stoc% solution !ill $ary depending
on the number of targets and the species under in$estigation. Please c+eck t+e la:el
on t+e tu:e for the exact concentration of the stoc% mixture. The follo!ing is only
intended as a rou&+ guide.
i" sin&le analytes-the detection antibody is usually supplied as a 5((x stoc% solution.
ii" .-; analytes-the detection antibody mixture is typically supplied as a '(x stoc% solution
"+u,an and ,ouse%( or a .'x stoc% solution "rat%.
iii" 5(<5; analytes-the detection antibody mixture is usually supplied as a .'x stoc% solution
"+u,an and ,ouse%.
i$" .(0 or ,ore analytes-the detection antibody mixture is usually supplied as a 5(x stoc%
solution +u,an and ,ouse%.
-" Prepare the appropriate !or%ing dilution of the detection antibody stoc% solution in
Bio-Plex Detection Anti:ody Diluent.
a" 5ocument all $olume calculations.
b" Protect the antibody from light.
c" @eep all antibody solutions on ice !hen not in use.
4" Remo$e the plate sealer and filter !ash /6 !ith 5(( l of Bio-Plex 7as+ Bu33er-
*" 9ently vortex the detection antibody and add .' l to each !ell.
B" 9o$er the !ells !ith plastic adhesi$e plate sealer( and blot the bottom of the plate.
7" 9o$er !ith foil and incubate for /( ,in at roo, te,perature !ith sha%ing.
a" /lo!ly ramp up to 505(( rp, and sha%e at high speed for /( sec
b" Reduce speed to /(( rpm for the remainder of incubation
PREPARE STREPTA28D8N-PE "D!R8N9 PRECEED8N9 8NC!BAT8ON AND
5( M8N PR8OR TO !SE%
'" 5etermine the total number of !ells that !ill be re>uired.
a" &s a precaution( add . extra !ells for e$ery ? !ells actually re>uired.
," 2i&orously vortex the /trepta$idin-P2 '00x" stoc% solution.
-" Prepare a '00 fold !or%ing dilution of the /trepta$idin-P2 '00x" stoc% solution in
Bio-Plex Assay Bu33er.
a" 5ocument all $olume calculations.
b" Protect the solution from light.
c" @eep on ice !hen not in use.
4" Remo$e the plate sealer and filter !ash /6 !ith 5(( l of Bio-Plex 7as+ Bu33er.
*" 2ortex the strepta$idin-P2 !or%ing dilution and add '( l to each !ell.
B" 9o$er the !ells !ith plastic adhesi$e plate sealer( and blot the bottom of the plate.
7" 9o$er !ith foil and incubate for 5( ,in at roo, te,perature !ith sha%ing.
a" /lo!ly ramp up to 505(( rp, and sha%e at high speed for /( sec
b" Reduce speed to /(( rpm for the remainder of incubation
?" Remo$e the plate sealer and filter !ash /6 !ith 5(( l of Bio-Plex 7as+ Bu33er.
A" Resuspend the beads in each !ell !ith 5.' l of Bio-Plex Assay Bu33er.
'0" 9o$er the !ells !ith plastic adhesi$e plate sealer( and thoroughly :lot the bottom of
the plate.
''" /ha%e the plate at 505(( rp, for /( sec = slo!ly ramp up to this high speed
'," Remo$e the sealer and read the plate.
Note) Set t+e Bio-Plex Reader to ready 5(( :eads1re&ion in a volu,e o3 '( u#
Bio-Plex Multiplex Cytokine Assay Dilution 7orks+eet
D of re>uired !ells EEEEEE
D of extra !ells EEEEEE , !ells for e$ery ? re>uired !ells"

EEEEEEE total number of !ells for dilution calculations
Bead Dilution
'( l1$ell
, l of anti-cyto%ine bead ,*x" stoc% solution0!ell
EEEEEEEE x , l F EEEEEEE of anti-cyto%ine bead ,*x" stoc% solution assay module"
EEEEEEEE x 4? l F EEEEEE of Bio-Plex Assay Bu33er A cyto%ine reagent %it"
EEEEEEEE x *0 l F EEEEEE total $olume
Detection Anti:ody Dilution
C4ote< The detection antibody solution is pro$ided either as a '00x( *0x( ,*x( or '0x
concentrate. Please c+eck t+e la:el on t+e tu:e 3or t+is in3or,ation. 5etection
antibodies for premixed panels containing , to A targets are typically supplied as a *0x
human( or mouse"( or ,*x rat" concentrate. 5etection antibodies for premixed panels
containing '0-'A targets are normally supplied as a ,*x concentrate human and mouse"(
!hereas detection antibodies for premixed panels containing ,0( or more targets are
supplied as a '0 x concentrate human and mouse". /ingle-plexes are generally supplied
as a '00x concentrate.
.' l1$ell
0.* l of detection antibody '(x" stoc% solution0!ell
EEEEEEEE x 0.* l F EEEEEEE of detection &b *0x" stoc% solution assay module"
EEEEEEEE x ,4.* l F EEEEEE of Bio-Plex Detection A: Diluent A cyto%ine reagent %it"
EEEEEEEE x ,* l F EEEEEE total $olume
or
.' l1$ell
' l of detection antibody .'x" stoc% solution0!ell
EEEEEEEE x ' l F EEEEEEE of detection &b ,*x" stoc% solution assay module"
EEEEEEEE x ,4 l F EEEEEE of Bio-Plex Detection A: Diluent A cyto%ine reagent %it"
EEEEEEEE x ,* l F EEEEEE total $olume
Streptavidin-PE Dilution
'( l1$ell
0.* l of strepta$idin-P2 '00x" stoc% solution0!ell
EEEEEEEE x 0.* l F EEEEEEE of strepta$idin-P2 '00x" stoc% solution cyto%ine reagent
%it"
EEEEEEEE x 4A.* l F EEEEEE of Bio-Plex Assay Bu33er A cyto%ine reagent %it"
EEEEEEEE x *0 l F EEEEEE total $olume