TITLE: PBMC surface flow staining
AUTHORS: cecilia , Alessandro Sette

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is about Flow staining.</div></div>

[STEPS]
?. [Day 1 - THAWING of PBMC]
Add  per donor vial being thawed to a labeled 50 ml tube.
[cell culture media]
?. [Day 1 - THAWING of PBMC]
Add  per 10 ml media of Benzonase to the 50 ml tube.
20 µl
?. [Day 1 - THAWING of PBMC]
Get as many vials of PBMC as needed.
?. [Day 1 - THAWING of PBMC]
Place the vials in  water bath to thaw – approximately .
37 °C
?. [Day 1 - THAWING of PBMC]
Before cells are completely thawed add them to the prepared 50ml tube.
?. [Day 1 - THAWING of PBMC]
Centrifuge tube with cells at .
Centrifuge: 1200 33, 7 min
?. [Day 1 - THAWING of PBMC]
Pour off media (cells in pellet) and resuspend in .
[cell culture media]
?. [Day 1 - THAWING of PBMC]
Take  and count cells (using trypan blue).
20 µl
?. [Day 1 - THAWING of PBMC]
Centrifuge tube with cells again at .
Centrifuge: 1200 33, 7 min
?. [Day 1 - THAWING of PBMC]
Pour off media and resuspend according to cell count to a final concentration of 10x106 cells per ml with HR5 media.DonorVialsCells/ml
(x106)Volume (mL)Total #
PBMCs (x106)Volume to reach 10x106% viability17
DonorVialsCells/ml
(x106)Volume (mL)Total #
PBMCs (x106)Volume to reach 10x106% viability17
?. [Day 1 - THAWING of PBMC]
Add  cells per well per plate (use 0.5 million instead of 1 million if necessary).123456789101112AAliquot 1BCAliquot 3 unstainedDEFGH
100 µl
123456789101112AAliquot 1BCAliquot 3 unstainedDEFGH
?. [Day 1 - THAWING of PBMC]
Spin plate , flick off liquid.
Centrifuge: 1400 33, 2 min, 4 10
?. [Day 1 - THAWING of PBMC]
Add , spin at , flick off liquid.
[cold PBS buffer]
Centrifuge: 1400 33, 2 min, 4 10
?. [Day 1 - THAWING of PBMC]
Resuspend cells in  (, ) for blocking.
[PBS with 10% FBS]
[PBS]
[FBS]
?. [Day 1 - THAWING of PBMC]
Incubate  at  (or in the fridge).
4 °C
?. [Day 1 - THAWING of PBMC]
Add  per well of stain mastermix (on top of the  so the total volume in each well becomes ).AntibodyAmount per tubeSTAIN MastermixCD4 APCEf7801 l ebio 470049422 lCD3 Alexa Fluor 7002 l BD 5579434 lCD8 BV6501µl Biolegend 3010422 lCD19 PECy71 l Tonbo 60-0199-T1002 lCD14 APC1 l Tonbo 20-0149-T1002 lCCR7 PerCPCy5.5BioLegend 3ul 3532206 lCD56 PE2ul Ebio 8025-0567-0254 lCD25 FITC2ul (large ab box) BD 5554314 lCD45RA eF450Ebio 1 l Ebio 48-0458-422 lLive/Dead AquaEbio (stored in freezer #11) 0.2ul0.5 lPBS buffer86µl per sample172 l
100 µl
[FBS]
200 µl
AntibodyAmount per tubeSTAIN MastermixCD4 APCEf7801 l ebio 470049422 lCD3 Alexa Fluor 7002 l BD 5579434 lCD8 BV6501µl Biolegend 3010422 lCD19 PECy71 l Tonbo 60-0199-T1002 lCD14 APC1 l Tonbo 20-0149-T1002 lCCR7 PerCPCy5.5BioLegend 3ul 3532206 lCD56 PE2ul Ebio 8025-0567-0254 lCD25 FITC2ul (large ab box) BD 5554314 lCD45RA eF450Ebio 1 l Ebio 48-0458-422 lLive/Dead AquaEbio (stored in freezer #11) 0.2ul0.5 lPBS buffer86µl per sample172 l
?. [Day 1 - THAWING of PBMC]
Incubate in dark in fridge .
?. [Day 1 - THAWING of PBMC]
Spin plate , flick off liquid.
Centrifuge: 1400 33, 2 min
?. [Day 1 - THAWING of PBMC]
Wash 2x with  per well of cold PBS Buffer.
100 µl
?. [Day 1 - THAWING of PBMC]
Resuspend in  and transfer to FACS tubes.
[PBS]
?. [Compensation controls with Beads]
Staining controlsHostConjugationBead/cellsμl/test/wellμl buffer/wellUnstained controlBeads100CD25MouseFITCBeads2 µl/test100CD56MousePEBeads2 µl/test100CCR7MousePerCPCy5.5Beads2 µl/test100CDI9PECy7Beads1 µl/test100CD45RAeF450Beads1 µl/test100CD19 instead of live/deadMouseV500Beads1 µl/test100CD8MouseBV650Beads1 µl/test100CD14MouseAPCBeads1 µl/test100CD3MouseAF700Beads2 µl/test100CD4MouseAPCEf780Beads1 µl/test100
Staining controlsHostConjugationBead/cellsμl/test/wellμl buffer/wellUnstained controlBeads100CD25MouseFITCBeads2 µl/test100CD56MousePEBeads2 µl/test100CCR7MousePerCPCy5.5Beads2 µl/test100CDI9PECy7Beads1 µl/test100CD45RAeF450Beads1 µl/test100CD19 instead of live/deadMouseV500Beads1 µl/test100CD8MouseBV650Beads1 µl/test100CD14MouseAPCBeads1 µl/test100CD3MouseAF700Beads2 µl/test100CD4MouseAPCEf780Beads1 µl/test100
?. [Compensation controls with Beads]
Add 1 drop beads (ebio Ultra comp beads).
?. [Compensation controls with Beads]
Spin down .
Centrifuge: 1400 33, 2 min, 4 10
?. [Compensation controls with Beads]
Add .
[PBS buffer and designated antibody]
?. [Compensation controls with Beads]
Incubate in the dark for  in fridge.
?. [Compensation controls with Beads]
Wash twice with cold PBS buffer.
?. [Compensation controls with Beads]
Resuspend to  and add to FACS tubes.
[PBS buffer]