Tepd, we advise employing a process of depleting dead cells (e.g., EasySepTM Dead Cell Removal (Annexin V) Kit) also as resting the cells prior to functional assessment. 13.four.2 Protocol for hepatic leukocyte staining–Reagents 1PBS LIVE/DEADTM Fixable Dead Cell Stain Kit Antibodies (see staining panels) Foxp3/Transcription Aspect Staining Buffer Set (or comparable) ddH2OEur J Immunol. Author manuscript; readily available in PMC 2020 July 10.SR-PSOX/CXCL16 Proteins Biological Activity 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 following 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 each effectively and centrifuge for five min at 500 g, discard supernatant For detection of surface molecules, prepare an Ab master mix in PBS and resuspend the cells in one hundred L Ab solution/wella,b Incubate for 30 min/4 in 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 100 L of Fixation/Perneabilization functioning solution per well, resuspend the cells, and incubate for 30 min at 4 within 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 10.Cossarizza et al.PageAdd 150 L Permeabilization Buffer/well and centrifuge for 5 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 can be maintain at four within the P-Selectin Proteins Gene ID darkAuthor Manuscript Author Manuscript Author Manuscript Author ManuscriptaTheuse of Ab master mixes is propose, these is usually ready either fresh ahead of the experiments or ready beforehand and stored at four inside the dark. Preparation beforehand need to be tested and validated against freshly prepared master mixes for each and every panel. The volume with the antibody master mix added might be modified determined by panel size or cell numbers.our practical experience, LIVE/DEAD Fixable Viability Dye’s is often added directly for the Ab master mix and stained simultaneously. Alternatively, an extra staining and washing step may be incorporated beforehand: For detection of death cells, prepare a live/dead staining resolution in PBS Add 50 L live/dead staining solution/well and re-suspend the cells Incubate for 30 min at 4 inside the dark Fill 150 L PBS/well and centrifuge for five min/500 g/4 ; discard supernatantbIncAlternativelyand depending on time-to-flow, we can propose fixing the cells with one hundred L four PFA for 20 min at four (or similar fixation reagents, e.g., BD CellFIXTM) prior to washing when and resuspending in 150 L PBS. Keep stained cells at four in the.