Tepd, we advise employing a process of depleting dead cells (e.g., EasySepTM Dead Cell Removal

Tepd, we advise employing a process of depleting dead cells (e.g., EasySepTM Dead Cell Removal (Annexin V) Kit) also as resting the cells just before functional assessment. 13.4.2 Protocol for hepatic leukocyte staining–Reagents 1PBS LIVE/DEADTM Fixable Dead Cell Stain Kit Antibodies (see staining panels) Foxp3/Transcription Factor Staining Buffer Set (or comparable) ddH2OEur J Immunol. Author manuscript; offered in PMC 2020 July 10.Cossarizza et al.PageEquipmentAuthor Manuscript Author Manuscript Author Manuscript Author Manuscript96-well microtiter plate, u- or v-bottom Centrifuge FCM tubes Flow cytometer BD LSR FortessaTM Laser: ultraviolet (355), violet (405 nm), blue (488 nm), green (561 nm), red (633 nm) Filter: 740/35, 380/14 for 355; 780/60, 710/40, 675/50, 610/20, 586/15, 525/50, 450/50 for 405; 710/40, 530/30, 488/10 for 488; 780/60, 670/30, 610/20, 586/15 for 561; 780/60. 730/45, 670/14 forProcedure Continued from 16.three.1 or soon after thawing of cryo-preserved samples Surface staining Transfer the cells into a 96-well microtiter (preferably u- or v-bottom) plate Centrifuge for five min/500 g/room temperature, discard supernatant Fill add 15000 L 1PBS to every single properly and centrifuge for 5 min at 500 g, discard supernatant For detection of surface molecules, prepare an Ab master mix in PBS and resuspend the cells in 100 L Ab solution/wella,b RORγ Inhibitor custom synthesis incubate for 30 min/4 within the dark Fill 15000 L PBS/well and centrifuge for five min/500 g/room temperature, discard supernatant Repeat the washing step Resuspend the cells in 150 L PBS/well and proceed to flow cytometric analysiscIntracellular stainingd Add one hundred L of Fixation/Perneabilization working remedy per well, resuspend the cells, and incubate for 30 min at four inside the darke Add 150 L1 Permeabilization Buffer/well and centrifuge for five min/500 g/ four ; discard supernatant Repeat the washing step Prepare the Ab solution for intracellular staining in Permeabilization Buffer and re-suspend the cells in 100 L Ab solution/well Incubate for 30 min at 4 inside the darkEur J Immunol. Author manuscript; offered in PMC 2020 July ten.Cossarizza et al.PageAdd 150 L Permeabilization Buffer/well and centrifuge for five min/500 g/4 ; discard supernatant Repeat the washing step Resuspend the cells in 150 L PBS/well and proceed to flow cytometric evaluation, Alternatively stained cells could be maintain at 4 inside the darkAuthor Manuscript Author Manuscript Author Manuscript Author ManuscriptaTheuse of Ab master mixes is advise, these may be ready either fresh just before the experiments or ready SIRT2 Activator Accession beforehand and stored at 4 inside the dark. Preparation beforehand must be tested and validated against freshly prepared master mixes for every single panel. The volume with the antibody master mix added may well be modified determined by panel size or cell numbers.our knowledge, LIVE/DEAD Fixable Viability Dye’s might be added straight for the Ab master mix and stained simultaneously. Alternatively, an added staining and washing step might be integrated beforehand: For detection of death cells, prepare a live/dead staining solution in PBS Add 50 L live/dead staining solution/well and re-suspend the cells Incubate for 30 min at four in the dark Fill 150 L PBS/well and centrifuge for 5 min/500 g/4 ; discard supernatantbIncAlternativelyand based on time-to-flow, we can advise fixing the cells with 100 L 4 PFA for 20 min at four (or equivalent fixation reagents, e.g., BD CellFIXTM) just before washing as soon as and resuspending in 150 L PBS. Retain stained cells at four in the.