For LSK lentiviral infection and rescue BMT, sorted LSK cells from either Hectd1f/f or Hectd1f/f;Vav (CD45.2) mice were cultured in SFEM media (StemCell Technologies Inc) supplemented with 10% FBS (SAFC Biosciences) and cytokines (100 ng/mL mSCF, 20 ng/mL mTpo, 20 ng/mL FLT3L, 20 ng/mL IL6) for 2 days. Lentivirus carrying mCherry/shLuc or mCherry/shmZNF622 were preloaded twice into a RetroNectin (T100B, Takara)-coated 12-well plate (Modlich et al., 2009 (link)). Cultured LSKs were transferred to the lentivirus-preload plates and incubated for one more day. At day3, 250k cultured LSKs were mixed with 500k Sca1-depleted competitor BM cells and injected into lethally-irradiated recipient mice. A small fraction of infected cells was spared for flow cytometry to evaluate the viral infection efficiency (Jiang et al., 2012 (link)).
Sfem media
Manufactured by STEMCELL
Sourced in Canada
SFEM media is a specialized cell culture medium designed to support the growth and maintenance of hematopoietic stem and progenitor cells. It is a serum-free, animal component-free formulation that provides the necessary nutrients and growth factors for the in vitro expansion and differentiation of these cells.
Automatically generated - may contain errors
Lab products found in correlation
Stem cell factor (scf), by Thermo Fisher Scientific (6 mentions)
Lsm 710 nlo, by Zeiss (2 mentions)
Ldh glo cytotoxicity assay, by Promega (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Retronectin t100b, by Takara Bio (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Interleukin 3 (il 3), by Thermo Fisher Scientific (1 mentions)
Hl 60, by American Type Culture Collection (1 mentions)
Penicillin streptomycin p s, by Corning (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Murine il 3, by Thermo Fisher Scientific (1 mentions)
Ba f3 cells, by Thermo Fisher Scientific (1 mentions)
Scf cytokines, by Thermo Fisher Scientific (1 mentions)
Ficoll paque plus, by GE Healthcare (1 mentions)
Rpmi 1640, by Corning (1 mentions)
Interleukin 6 (il 6), by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Ba f3 cells, by Leibniz Institute DSMZ (1 mentions)
Pspax2, by Addgene (1 mentions)
Cellrox deep red reagent, by Thermo Fisher Scientific (1 mentions)
9 protocols using sfem media
1
Lentiviral Infection and Rescue BMT
2
Hydrogel Differentiation Patterns of HSPCs and MSPCs
3
Maintaining Diverse Leukemia Cell Lines
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U937, HL-60, and 293T cells were purchased from ATCC. Ba/F3 cells were purchased from DSMZ. THP-1, MV4-11, and MOLM-14 cells were provided by Scott Armstrong. MOLM-13 cells were provided by Benjamin Ebert. All cell lines were maintained in RPMI 1640 (Cellgro) supplemented with 1% penicillin/streptomycin (PS; Cellgro) and 10% FBS (Sigma-Aldrich) at 37°C with 5% CO2. The 293T cells were maintained in Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 10% FBS (Invitrogen) and 1% PS. Ba/F3 cells were maintained in RPMI-1640 supplemented with 1% PS, 10% FBS, and IL-3 (PeproTech).
Primary patient AML blasts were collected from peripheral blood or bone marrow aspirate after obtaining informed consent under Dana-Farber Institute Internal Review Board–approved protocols. Mononuclear cells were isolated using Ficoll-Paque Plus (GE Healthcare), and red blood cells were lysed (eBioscience). AML blasts from patients were maintained in SFEM (Stemcell Technologies) with SCF, FLT3L, IL-3, IL-6, and GCSF (PeproTech). For drug treatment of primary AML cells, RPMI-1640 with PS and FBS were used.
Murine MLL-AF9–expressing leukemia cells were obtained from a quaternary bone marrow transplant model from Benjamin Ebert’s laboratory. Cells were maintained in SFEM media (Stemcell Technologies) with murine IL-3, IL-6, and SCF cytokines (PeproTech).
Primary patient AML blasts were collected from peripheral blood or bone marrow aspirate after obtaining informed consent under Dana-Farber Institute Internal Review Board–approved protocols. Mononuclear cells were isolated using Ficoll-Paque Plus (GE Healthcare), and red blood cells were lysed (eBioscience). AML blasts from patients were maintained in SFEM (Stemcell Technologies) with SCF, FLT3L, IL-3, IL-6, and GCSF (PeproTech). For drug treatment of primary AML cells, RPMI-1640 with PS and FBS were used.
Murine MLL-AF9–expressing leukemia cells were obtained from a quaternary bone marrow transplant model from Benjamin Ebert’s laboratory. Cells were maintained in SFEM media (Stemcell Technologies) with murine IL-3, IL-6, and SCF cytokines (PeproTech).
4
Oxidative Stress Analysis in LSK Cells
5
Cytotoxicity Evaluation of GelMA Hydrogels
6
Isolation and Culture of AML Cells
7
Lentivirus Production and Concentration
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293x cells were transfected using polyethylenimine (Polysciences Cat# 24765-2) with the packaging plasmids pMD2.G (Addgene ID: 12259) and psPAX2 (Addgene ID: 12260), and gRNA-containing plasmids. Lentiviruscontaining supernatant was collected 72 hours post-transfection and spun at 854g for 5 minutes to remove cells. The supernatant was filtered through a 45 μm low-protein binding filter (Whatman Cat# EW-29705-54), placed in ultracentrifuge tubes (Beckman Coulter Cat #344058), and spun at 20,000 RPM (71,934.8g) at 4°C for 2 hours in a Beckman Coulter ultracentrifuge with an SW28 rotor. Concentrated lentivirus was then resuspended in SFEM media (STEMCELL Technologies Cat# 9600) overnight before freezing at -80°C. Lentivirus was titered on 293x cells to determine viral particles per mL.
8
Oxidative Stress Quantification in LSK Cells
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Purified LSK cells were encapsulated in either 5 wt% GelMAL (peptide) or GelMA (PI) at 5x10 5 LSK/mL and cultured for 2 h at 5% CO2 and 37 °C in 500 µL SFEM media (#09650, Stemcell Technologies) supplemented with 100 ng/mL SCF (#250-03, Peprotech) and 1% PenStrep.
After 2 h, hydrogels were resuspended in 5 μM CellROX Deep Red Reagent, for oxidative stress detection (#C10422, ThermoFisher Scientific) in media and incubated for 1 h. Hydrogels were washed in PBS for 5 min and fixed in 4% formaldehyde for 15 mins. Hydrogels were washed in PBS and stained with Hoescht 33342 (1:2000) before imaging. A negative control was created with no CellROX reagent (Figure S2). Z-stack images were captured using a Zeiss LSM 710 NLO (Zeiss, Oberkochen, Germany; 40x W objective). Maximum intensity images were processed using CellProfiler to quantify the mean CellROX fluorescence in each cell, with corrected fluorescence intensity reported as the fluorescence intensity with background (three reference spots) removed. A total of 4 mice were used to create n = 3 samples per hydrogel condition, with at least 10 cells per hydrogel (n = 30 cells per condition) and 3 reference spots per image.
After 2 h, hydrogels were resuspended in 5 μM CellROX Deep Red Reagent, for oxidative stress detection (#C10422, ThermoFisher Scientific) in media and incubated for 1 h. Hydrogels were washed in PBS for 5 min and fixed in 4% formaldehyde for 15 mins. Hydrogels were washed in PBS and stained with Hoescht 33342 (1:2000) before imaging. A negative control was created with no CellROX reagent (Figure S2). Z-stack images were captured using a Zeiss LSM 710 NLO (Zeiss, Oberkochen, Germany; 40x W objective). Maximum intensity images were processed using CellProfiler to quantify the mean CellROX fluorescence in each cell, with corrected fluorescence intensity reported as the fluorescence intensity with background (three reference spots) removed. A total of 4 mice were used to create n = 3 samples per hydrogel condition, with at least 10 cells per hydrogel (n = 30 cells per condition) and 3 reference spots per image.
9
Cytotoxicity Evaluation of Hydrogels
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5 wt% GelMAL-peptide and 5 wt% GelMA hydrogels containing 10 5 LSK/mL were immediately placed in 500 µL SFEM media (#09650, Stemcell Technologies) supplemented with 100 ng/mL SCF (#250-03, Peprotech, Rocky Hill, NJ) and 1% PenStrep and cultured at 5% CO2 and 37 °C. At time points of 1 h and 48 h samples were tested for cytotoxicity via a commercially available LDH-Glo™ Cytotoxicity Assay (#J2380, Promega, Madison, WI). In brief, 10 µL conditioned media was diluted in LDH Storage Buffer (200 mM Tris-HCl, 10% glycerol, 1% BSA, pH 7.3) and reacted with LDH Detection Reagent on a shaker for 1 h at RT. Immediately following the reaction, the samples were moved to an opaque-walled 96-well plate in triplicate and luminescence was read using a BioTek Synergy HT Plate Reader and Gen5 Software (BioTek Instruments, Inc., Winooski, VT). Relative luminescence was calculated by subtracting the average values from a background control (media). Values of each sample are reported as the mean of each triplicate, and n = 4 (GelMAL-peptide) and n = 5 (GelMA).
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