70 um cell strainer

Single-cell suspensions were prepared from spleen and thymus by passing cells through a 70 um cell strainer (Falcon). Bone marrow cells from tibias, femurs and spine were crushed with PBS +2% FBS and filtered. After cell counting, cells were stained with following antibody (BD Biosciences, eBiosciences) combinations: Biotin-conjugated lineage marker cocktail (Ter-119, Gr-1, Mac-1, B220, CD4, CD8), HSC (Sca1, c-Kit, CD34, Flt3), T lymphocytes (CD4, CD8, CD25, CD44, Ter119), B lymphocytes (B220, CD4, CD8, CD19, CD43). For KSL cells sorting, cKit positively selected cells were used for staining. Briefly, cells were stained with c-Kit-APC antibody for 30 min on ice, washed with PBS and incubated with anti-APC microbeads for 15 min at room temperature. c-Kit positive cells were collected using a MACS separation column and used for HSC staining. FACS analysis was carried out on LSRII (BD Biosciences) and Gallios (Beckman coulter) at the MDACC Flow Cytometry and Cellular Imaging Core Facility. FACS data was analyzed with Flowjo software V10.0.8.

Spleens were dissociated by mechanical disruption and digested with collagenase/dispase (20 ug/ml, Roche, Indianapolis, IN) and DNAse I (300 ug/ml, Roche) for 30 min at 37°C and passed through a 70 um cell strainer (BD Biosciences, San Jose, CA). Heparinized blood was collected via cardiac puncture and RBCs were removed by dextran sedimentation. Remaining RBCs were lysed with ammonium chloride. Cells were stained at 4°C in PBS with Live/Dead Violet Fixable Stain kit (Invitrogen, Carlsbad, CA), washed, then stained in PBS with 0.5 mM EDTA, 0.2% BSA, 0.09% azide, and 2% normal rat serum (Jackson ImmunoResearch, West Grove, PA). Anti-mouse CD45 (30-F11) APC, anti-mouse CD11b (M1/70) PerCpCy5.5 (eBioscience, San Diego, CA), anti-mouse Ly6C (AL-21) PE, anti-mouse CCR2 (475301) Fluorescein (R&D Systems, Minneapolis, MN), and anti-mouse Ly6G (1A8) PE-Cy7 antibodies were purchased from BD Biosciences except as indicated. Anti-Rat/Hamster CompBeads (BD Biosciences) were used to set compensation. Data were collected on an LSRII cytometer (BD Biosciences) and analyzed with FlowJo 7.6.1 (TreeStar, Ashland, OR).

Blood and spleen samples from RP mice or controls were lysed by red blood cell (RBC) lysis solution (Biolegend, San Diego, CA). Cells were then passed through 70 um cell strainer (BD Biosciences, Franklin Lakes, NJ) to prepare monolayer cell suspension. Prolapsed or normal rectal tissues were enzymatically digested with enzyme cocktail containing collagenase IV (Worthington Biochemical Corp, Lakewood, NJ), dispase (Gibco), and DNAse I (Roche). Digested cell suspensions were subjected to 40% versus 80% percoll gradient centrifugation (GE Healthcare, Pittsburgh, PA) to enrich leukocytes. To phenotypically analyze immature myeloid cells, cells from blood, spleen, or RP tissue were stained with the following antibodies: PEcy7-CD45 (clone 30-F11), APC-CD11b (clone M1/70), and Percp-Cy5.5-Gr1 (clone RB6-8C5). Multicolor flow cytometry was performed on LSRII flow cytometer (BD Biosciences). All flow antibodies were obtained from Biolegend. Data were analyzed and presented by using Flowjo 9 software (Tree Star, Ashland, OR).

Mice were anesthetized by inhalation of 10% Isoflurane in mineral oil (v/v) and then retro-orbitally injected with 2.5 μg of anti-CD45-APC-Cy7 antibody (clone 30-F11, Biolegend) in 200 μl of PBS. Three minutes after antibody injection mice were euthanized by Isoflurane inhalation and cervical dislocation. Lungs, MLN, peripheral LN, BM and blood were collected from the mice depending on the experiment. Solid tissues were placed in complete media (RPMI1640, 5% FCS, 10 mM HEPES, 1x NEAA, 1 mM Sodium Pyruvate, 55 μM 2-mercaptoethanol) containing 1 mg/ml type 2 collagenase and 30 μg/ml DNase, minced with scissors and incubated for 30 minutes at 37° C. Tissues were then forced through a 70-uM cell strainer (BD) and washed twice in collagenase wash buffer (PBS + 2% FBS (v/v), 2 mM EDTA). Mouse femur and tibia leg bones were cleaned of tissue before removing their ends with scissors. Marrow was then extracted by perfusing with 5 mL of complete media using a 27G needle. Blood was collected in complete media. RBC were removed from all tissues using ACK lysis buffer (155 mM NH₃Cl, 10 mM KHCO₃, and 88 μM EDTA) and live cells were counted using trypan blue exclusion and a Countess cell counter (Thermo Fischer Scientific)

The different cell types cultured in the presence of absence of Dox (0.1 μg/ml) were resuspended in PBS+FBS+EDTA buffer (FACS buffer) and filtered through a 70 um cell strainer (BD Biosciences, Bedford, MA). Live cells identifier by 7-AAD viability dye exclusion were analyzed for eGFP expression using a FACS Canto II flow cytometer equipped with the FACS Diva analysis software (BD Bioscience).

Flow cytometry analysis and MACS purification were performed as previously described^{24 (link)} with the following modifications. Cells were dissociated into a single-cell suspension by incubating in accutase for ~45 min. The single-cell suspension was then passed through a ~70 uM cell strainer (BD Biosciences), washed, and resuspended in Live Cell Imaging Solution (ThermoFisher Scientific) for analysis with an SH800S Cell Sorter (Sony Biotechnology, San Jose, CA). For flow analysis, BSC and FSC were used to select and subset live cells, and only live cells were used to quantify a number of tdTomato⁺ cells. A gate was set up using WT hES cells differentiated to day 95. Since it is not practical to differentiate a WT line every time a reporter line is differentiated, the same gate was used for every flow analysis. For MACS purification, cells were resuspended in MACS buffer after passing through the cell strainer. A CD90.2 (THY1.2), O4, or A2B5 MicroBeads were added to the cell suspension and incubated at room temperature for 15 min for cell binding. Cells were generally run through the MS column twice without additional supplementation of MicroBeads to increase the purity and achieve ~90% tdTomato⁺ cells. All MACS reagents were purchased from Miltenyi Biotec (Auburn, CA) and manufacturer instructions were followed.