Sa3800

The level of core 1 structure on cell surface was measured by flow cytometry with mouse mAb 3C9 (an in-house produced antibody) specific to core 1 glycosylation^{69 (link)}. Cells were incubated on ice with 3C9 mAb (undiluted hybridoma supernatant which is equivalent to 1:1 dilution) for 30 min, followed by washing and incubation with Alexa Fluor 647 conjugated goat anti-mouse IgM (1 µg/mL) (Invitrogen, catalogue: A21235) for 30 min. Diluting and washing was performed in PBS with 1% BSA and cells were resuspended for flow cytometry analysis (SONY SA3800). Mean fluorescent intensity of the binding of mAb 3C9 populations was quantified by FlowJo software (FlowJo LLC).

Cells were washed with 1xPBS (Sigma, Louis, MO), then surface stained by incubating with antibodies for 30 min at 2 ~ 8 °C. They were subsequently washed again prior to flow analysis on Sony SA3800. Anti-CD4 (clone OKT4), anti-CD8a (clone RPA-T8), anti-PD-1 (clone EH12.2H7), anti-TIM3 (clone F38-2E2), anti-LAG3 (clone 7H2C65), anti-CD3 (clone UCHT1 or clone HIT3a), anti-CD62L (clone DREG-56), anti-CD45RO (clone UCHL1), anti-CD69 (clone FN50), anti-CD137 (clone 5F4), anti-CD25 antibodies (clone BC96), and Streptavidin were purchased from Biolegend (San Diego, CA). After collecting the supernatant, protein purification was accomplished with a protein-A affinity Beads. Biotin were labeled to purified CD70 protein with kit.

Cells were grown overnight in YPD at 30˚C, and the following morning, saturated cultures were diluted to A₆₀₀ ≍ 0.2 and grown until mid-log phase, A₆₀₀ ≍ 0.6. Cells were then washed in PBS, resuspended in YPD + 300-mM hydroxyurea, and placed at room temperature for 60 minutes with agitation. Arrested cells were then washed 2 × in fresh YPD and then resuspended in YPD, and placed at 30˚C for outgrowth after HU arrests. We took aliquots of the cell suspension at various timepoints for analysis. For DNA content analysis, cells were first crosslinked with 0.5% paraformaldehyde for 15 minutes at 4˚C. After 2 × washes with PBS, crosslinked cells were then resuspended in ice-cold (−20˚C) 70% methanol and incubated at 4˚C for 1 hour. Cells were then washed with 2 × PBS, resuspended in PBS + 2.5-µM SytoxGreen, and incubated at 30˚C for 30 minutes. For analysis of HTA-mNeonGreen, aliquots of cells were taken at the appropriate timepoints, washed 2 × in ice-cold PBS, and placed on ice until analyzed for flow cytometry. Cells were then analyzed using a spectral cell analyzer (Sony SA3800), and data from approximately ∼30,000 events were analyzed in the FlowJo software.

The following monoclonal antibodies (mAbs) and their isotype controls were used for flow cytometry: APC human PD-1, APC human PD-L1, Brilliant Violet human PD-L1, APC human CD86, APC human CD11b, PE human CD247 (CD3z), APC mouse CD11b; PE human EGFR from R&D Systems; APC mouse PD-L1 from Tonbo Biosciences. In addition, following reagents were used in flow cytometry: recombinant human PD-L1 Fc chimera Biotin protein (R&D Systems); APC Streptavidin (Tonbo Biosciences); Zombie NIR viability dye from BioLegend; CellTrace Violet, CellTrace CFSE, and CellTrace Yellow from Invitrogen.
For flow cytometry analysis, cells were resuspended in Cell Staining Buffer (BioLegend) containing Fc receptor blocker (Miltenyi Biotec) and incubated for 10 minutes at 4°C. Then the cells were stained in PBS containing 1:1000 diluted Zombie NIR viability dye (BioLegend) for 30 minutes at room temperature. Finally, the cells were stained with antibodies diluted in Cell Staining Buffer for 20 minutes at 4°C. Cells were washed twice with Cell Staining Buffer between the staining steps and once prior to data acquisition using SONY SA3800 or SONY iD7000 spectral flow cytometer. The flow cytometry data was analyzed using FlowJo software.

Serum anti-KEL IgM, IgG, IgG1, IgG2b, IgG2c, and IgG3 were measured by flow cytometric crossmatch. Anti-KEL IgM was measured 5 days after transfusion, and anti-KEL IgG was measured 7, 14, 21, and 28 days after transfusion as previously described (40 (link)). Briefly, serum from transfused mice was incubated with K1 or C57BL/6 RBCs and subsequently stained for RBC-bound IgM or IgG (goat anti-mouse IgM FITC or goat anti-mouse IgG APC). Secondary antibodies for IgG subsets included goat anti-mouse IgG1 PE, goat anti-mouse IgG2c APC, goat anti-mouse IgG2b FITC, and goat-anti mouse IgG3 BV421 (Jackson ImmunoResearch, West Grove, PA, United States). The adjusted MFI was calculated by subtracting the reactivity of serum with syngeneic C57BL/6 RBCs from the reactivity of serum with K1 RBCs. Graphed anti-KEL IgG data represents the peak antibody response, 21–28 days following transfusion. Flow cytometry of RBCs was performed using a SONY SA 3800 (San Jose, CA, United States) or a Cytek Northern Lights 3000 spectral analyzer (Fremont, CA, United States) and analyzed using FlowJo software (Tree Star, Ashland, OR, United States).