Lsrii analyzer

GFP and RFP-expressing cell lines were obtained by transforming cells with pTX-GFP (Dictybase ID: 11)
^{17 (link)} or pTX-RFP (Dictybase ID: 112) plasmids using a standard electroporation procedure. Cells were grown in 75cm
² flasks until dense but not confluent (usually 1 day before confluency). The medium was changed 4–6h before transformation. For transformation cells were re-suspended in 10mL of ice-cold HL5 and kept on ice for 30min. Cells were centrifuged for 5min, 500 g at 4°C. Supernatant was re-suspended in 800μl of electroporation buffer and transferred into ice cold 4mm electroporation cuvettes containing 30μg of plasmid DNA. Cells were electroporated at 0.85 kV and 25 mF twice, waiting for 5 s between pulses. Cells were transferred from the cuvette to 75cm
² flask with HL5. The next day, transformants were selected with 5μg/ml G418 (Sigma-Aldrich). The concentration of G418 was gradually increased to 20μg/ml G418 over 1–2 weeks. Transformed strains were maintained at this concentration of G418, yielding GFP and RFP-expressing cell lines that were analyzed by flow cytometry on a Becton-Dickinson LSRII analyzer to confirm their unimodal cellular fluorescence distribution (>99% of fluorescent cells upon analysis of 10
⁶ cells, see
Supplementary Figure S6).

The cell samples used for FACS assays were prepared according to the previously reported protocol^{37 (link)}. MCF-7 and SKMEL-28 cancer cells were incubated in ATCC-formulated EMEM and ATCC-formulated DMEM, both containing 10% FBS and 1% antibiotics. After being treated with trypsin-EDTA solution, the cells were harvested and washed with FACS buffer (PBS containing 5% FBS). The cells were incubated with 50 µL of normal mouse serum (1:10 dilution) or a day 38 pooled antisera (1:10 dilution) at 4 °C for 30 min. Subsequently, the cells were washed and treated with Dylight 633-linked goat anti-mouse kappa antibody (2 µL in 50 µL FACS buffer) at 4 °C for 30 min. The labeled cells were washed, suspended in the FACS buffer, and finally analyzed with a Becton Dickinson LSR II Analyzer.

Globo H-expressing MCF-7 and globo H-negative SKMEL-28 cell lines were used in the experiments. MCF-7 cell was incubated in ATCC-formulated Eagle's Minimum Essential Medium (EMEM) containing 10% FBS and 1% antibiotics, and SKMEL-28 cell was incubated in ATCC-formulated DMEM containing 10% FBS and 1% antibiotics. Both were harvested after treatment with trypsin–EDTA solution. Cells (about 1.0 × 10⁶) were washed twice with FACS buffer (PBS containing 5% FBS) and incubated with 50 μL of normal mouse serum (1 : 10 dilution) or a day 38 pooled antiserum (1 : 10 dilution) at 4 °C for 30 min. Thereafter, the cells were washed again with FACS buffer and incubated with FITC-linked goat anti-mouse kappa antibody (2 μL in 50 μL FACS buffer) at 4 °C for 30 min. Finally, cells were washed and suspended in 0.8 mL of FACS buffer for FACS analysis on a Becton Dickinson LSR II Analyzer at the Microscopy, Imaging and Cytometry Resources Core, Wayne State University.

Cells were grown to confluence, dissociated, and pelleted for resuspension in 50 μL of staining media (1M HEPES (pH 7.2), 1M NaN₃, 2% fetal bovine serum in Hank's Buffered Salt Solution). The cell suspension was gently mixed with 1 mL 80% ethanol. The cells were centrifuged at 200 x g for 5 min at 4°C for pelleting and resuspended in Hank's Buffered Salt Solution (HBSS, Wisent Inc.) containing 2 mg/mL RNAse A (Qiagen Inc., Toronto, ON, Canada) for 5 minutes at room temperature. Cells were then pelleted and resuspended in HBSS containing 0.1 mg/mL propidium iodide and 0.6% (w/v) NP40 at room temperature for 30 minutes. Subsequently, the cell pellet was resuspended in 500 μL of staining media and filtered through a 100 μm nylon cell strainer (BD Biosciences Discovery Labware, Bedford, MA, USA). A total of 10,000 cells were sorted for each cell line and experimental condition and each experiment was performed in triplicate. An LSR II analyzer (Becton Dickinson, Franklin Lakes, NJ, USA) was used with FACSDiva software. The excitation wavelength was 523 nm and the emission filter was LP600, BP610/20. Data analysis was conducted with FlowJo (Tree Star Inc., Ashland, OR, USA). Doublets were excluded by gating using the PI-intensity versus FSC-W graph as described previously.[4 (link)]

Mice were anesthetized with isoflurane (2.5% v/v induction, 1.5% v/v maintenance), and perfused with 10 ml of ice‐cold saline. De‐capsulated kidneys were minced and digested in Liberase DL (Roche) in Dulbecco's Modified Eagle's Medium, 10 mM HEPES, pH 7.4 at 37°C for 30 min while shaking. Ice‐cold isolation buffer (1% v/v BSA, 2 mM ethylenediaminetetraaceticacid [EDTA] in 1× PBS) was added to stop the enzymatic reaction. Tissues were disaggregated using 18‐G and 20‐G needles, followed by passage through a 40 μm filter and centrifuged. Red blood cells were lysed using ACK lysis buffer. Kidneys were washed in staining buffer (0.5% BSA, 0.01% sodium azide, 1× PBS) followed by Fcγr2/3 block (Clone 93) and subsequent staining for myeloid and lymphoid cells. AccuCheck beads (Life Technologies) were used to determine absolute numbers of cells by tissue mass normalization. Flow cytometry antibodies are listed in Table 1. 7‐Aminoactinomycin was used to exclude dead cells. Flow cytometry data were collected on a Becton‐Dickenson LSRII analyzer and data were analyzed using FlowJo (TrecStar Software).

The procedure was conducted as previously described [32 (link)] with slight modification. Briefly, the blood cell pellet was stained with LIVE/DEAD Aqua for 10 min at rt followed by washing with 500 µL flow buffer. Cells were stained with antibody cocktails with the following fluorochrome-conjugated antibodies for 30 min at rt: Alexa Fluor 488-labeled anti-CD20 (clone 2H7); FITC-labeled anti-CD56 (clone HCD56); Pacific Blue-labeled anti-CD15 (clone W6D3); APC-labeled anti-CD3 (clone UCHT1); Brilliant Violet 785-labeled anti-CD14 (clone M5E2); Brilliant Violet 605-labeled anti-CD11b (clone ICRF44). BD lysing solution was then added to lyse red blood cells and fix other nucleated cells for 30 min at rt. Cells were washed with wash buffer (PBS with 0.1% NaN₃) twice and resuspended in 200µL flow buffer. Cells were analyzed on a Becton Dickinson LSRII Analyzer at Stanford Shared FACS facility. Data were analyzed using FlowJo version 10 (FlowJo LLC, Ashland, OR, USA).