25-0719-41

antibody from Invitrogen Antibodies

Targeting: TFRC CD71, p90, TFR1

Provider product page for 25-0719-41

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Antibody Data
Antigen structure
References [25]
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Flow cytometry [1]
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Product number: 25-0719-41 - Provider product page
Provider: Invitrogen Antibodies
Product name: CD71 (Transferrin Receptor) Monoclonal Antibody (OKT9 (OKT-9)), PE-Cyanine7, eBioscience™
Antibody type: Monoclonal
Antigen: Other
Description: Description: The OKT9 monoclonal antibody reacts with human CD71, a 170-180 kDa type II transmembrane protein. CD71, the transferrin receptor, exists as a homodimer on the cell surface and is essential for cellular growth. CD71 is expressed by immature proliferating cells and at low levels on resting mature lymphocytes. Antigen or mitogen stimulation of T and B cells upregulates the expression of CD71. Applications Reported: This OKT9 (OKT-9) antibody has been reported for use in flow cytometric analysis. Applications Tested: This OKT9 (OKT-9) antibody has been pre-titrated and tested by flow cytometric analysis of stimulated normal human peripheral blood cells. This can be used at 5 µL (0.5 µg) per test. A test is defined as the amount (µg) of antibody that will stain a cell sample in a final volume of 100 µL. Cell number should be determined empirically but can range from 10^5 to 10^8 cells/test. Light sensitivity: This tandem dye is sensitive to photo-induced oxidation. Please protect this vial and stained samples from light. Fixation: Samples can be stored in IC Fixation Buffer (Product # 00-822-49) (100 µL cell sample + 100 µL IC Fixation Buffer) or 1-step Fix/Lyse Solution (Product # 00-5333-54) for up to 3 days in the dark at 4°C with minimal impact on brightness and FRET efficiency/compensation. Some generalizations regarding fluorophore performance after fixation can be made, but clone specific performance should be determined empirically. Excitation: 488-561 nm; Emission: 775 nm; Laser: Blue Laser, Green Laser, Yellow-Green Laser. Filtration: 0.2 µm post-manufacturing filtered.
Reactivity: Human
Host: Mouse
Isotype: IgG
Antibody clone number: OKT9 (OKT-9)
Vial size: 25 Tests
Concentration: 5 µL/Test
Storage: 4° C, store in dark, DO NOT FREEZE!

TFRC protein structure - 25-0719-41 shown in red.

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Experimental details: Fig. 5 Derivation of TFRC cell clones. a Outline of the clone-derivation protocol used for TFRC and MALAT1 knock out cells, indicating approximate time required. b First and c second stage PCRs to genotype clones. Primer combination schemes are indicated below the electrophoresis gels. H: TFRC_B cell clone genotyped as heterozygote; WT, cell clones genotyped as wild type; +, positive control wild type cells; H 2 O, water. d qRTPCR for TFRC mRNA, normalised to GAPDH. Error bars indicate the standard deviation of three technical replicates. e Flow cytometry analysis of surface levels of TFRC protein. Left : histogram of cell fluorescence intensity counts. Right : Calculation of relative stain index. f Sequencing analysis of mutant junction of the heterozygous clones. In red, region complementary to the gRNA variable region; Green, PAM sequences; Blue, indel. Expected cut location is marked with vertical bar

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Supportive validation

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Experimental details: Figure 3 Activation of KLF1 at day 10 of differentiation results in enhanced erythroid differentiation of hiPSCs. (A) : Schematic of the pZDonor-AAVS1 Puro-CAG-HA-KLF1-ER T2 -PA construct. (B) : Western blot analyses of nuclear cell lysates from adult CD34 + cells that had been differentiated for 6 days into erythroid progenitors, control undifferentiated and differentiated (day 10) induced pluripotent stem cells (iPSCs), undifferentiated iKLF1.2 iPSCs and iKLF1.2 iPSC that had been differentiated for 10 days then treated with tamoxifen for 3 hours. Endogenous KLF1 and the expected larger sized KLF1-ER T2 fusion protein was detected with the anti-KLF1 antibody and the anti-Lamin B1 antibody was used to detect nuclear proteins as a loading control. (C) : Flow cytometry analysis using antibodies against CD235a and CD71 of cells present at day 15 of the erythroid differentiation protocol in control iPSCs and iKLF1.2 iPSC cell lines in the presence (+) and absence (-) of tamoxifen from day 10. ( D) : Quantitation of flow cytometry data representing three independent experiments. Error bars represent SEM. p values were calculated using one-way ANOVA followed by Holm-Sidak's multiple comparison test (* p < .05). ( E) : Image showing the cell pellets from one representative experiment demonstrating a smaller but more intense red pellet in the tamoxifen-treated iKLF1.2 cell line. Abbreviation: iPSCs, induced pluripotent stem cells.

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Experimental details: Fig. 4 Sequence deletion in bulk cells. a Outline of the genomic PCR (gPCR) primer strategy used for genotyping. b Deletion of TFRC promoter (construct B in Fig. 2 ), as validated by electrophoresis of gPCR products. Wild type gDNA and water templates are used as positive and negative controls, respectively. Green and red arrows indicate the size of PCR products expected from wild type (WT) and deleted alleles. Note that in this and subsequent panels, separated lanes originate from the same original agarose gel, rearranged for clarity. c - e gPCR on bulk cells transfected with the indicated DECKO plasmids targeting ( c ) TFRC promoter, ( d ) MALAT1 upstream promoter, ( e ) MALAT1 major promoter. ( f - h ) qRTPCR on cell samples shown in ( c - e ). Control indicates RNA from cells transfected with a DECKO targeting GFP. Levels were normalised to GAPDH. Error bars show the standard deviation of three technical replicates. i Expression of TFRC protein on cell surface, as determined by flow cytometry analysis of antibody-stained cells. Left : histogram of cell fluorescence intensity counts. Right : Calculation of relative stain index, a normalised measure of fluorescence intensity

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Experimental details: Fig. 5 Derivation of TFRC cell clones. a Outline of the clone-derivation protocol used for TFRC and MALAT1 knock out cells, indicating approximate time required. b First and c second stage PCRs to genotype clones. Primer combination schemes are indicated below the electrophoresis gels. H: TFRC_B cell clone genotyped as heterozygote; WT, cell clones genotyped as wild type; +, positive control wild type cells; H 2 O, water. d qRTPCR for TFRC mRNA, normalised to GAPDH. Error bars indicate the standard deviation of three technical replicates. e Flow cytometry analysis of surface levels of TFRC protein. Left : histogram of cell fluorescence intensity counts. Right : Calculation of relative stain index. f Sequencing analysis of mutant junction of the heterozygous clones. In red, region complementary to the gRNA variable region; Green, PAM sequences; Blue, indel. Expected cut location is marked with vertical bar

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Experimental details: Fig. 5 Characterization of in vitro-differentiated human RBCs. a The proliferation curve of human RBCs. ( n = 3, mean +- S.E.M.). b Differentiating cells were characterized by flow cytometry for c-kit, CD71, CD235A, and Hoechst at the end of each culture stage. c Giemsa and hemoglobin staining of human RBCs at the end of each culture stage. d In vitro-differentiated human RBCs circulate for up to 7 days in macrophage-depleted NOD/SCID mice. Five hundred million six-stage cultured RBCs were labeled with CFSE and injected intravenously into NOD/SCID mice that have been treated with clodronate liposomes. The recipient mice were then bled at the indicated time points as indicated for further flow cytometric analyses. The transfused human RBCs were identified by tracing CFSE and CD235A expression

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Experimental details: Figure 5 CD47, CD71, and EpCAM Are Expressed on Ovarian Cancer-Derived EVs (A) Flow cytometry of the beads covered with IgG and antibodies against CD71, CD47, PALP, or MUC18 incubated with the medium conditioned by Kuramochi cells (palmtdTomato positive) (charts representative of three experiments). (B) Fraction of palmtdTomato-positive beads depending on CD47 and CD71 expression on EVs from the panel of five cell lines (n = 3). (C) mbGluc activity from beads with EVs captured by antibodies against MUC18, CD47, and CD71 from conditioned medium of Kuramochi cells (following removal of cells; n = 3). (D) Fraction of palmtdTomato-positive beads depending on antigen expression on EVs from DF30 and OV90 cell lines (n = 3). (E) Diagram of in vivo experiment to measure capture of EVs from serum of OVCAR5 tumor-bearing animal (left) and mbGluc activity from beads with EVs depending on the antigen expression (right) (n = 3 animals, two serum samples were processed from each animal, bioluminescence read in three to five technical replicates). (F) EVs were isolated by differential centrifugation and filtration steps from ascites from patients with ovarian cancer (n = 3) and with benign conditions such as cirrhosis or hepatitis (n = 3). Proteins were resolved by SDS-PAGE and stained with antibodies to EpCAM, CD63, and B(beta)-actin and detected with chemiluminescence. See also Figure S3 .

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Experimental details: Figure 1. An Epigenomic and Transcriptional Time Course of Human Erythroid Differentiation (A) Schematic outline of generating matched transcriptome and open chromatin profiles across human erythropoiesis. (B) FACS gating scheme showing expression of surface markers CD71, CD235a, CD49d, and BAND3 used to sort indicated populations P1-P8 (P1, MyP; P2, CFU-E; P3, ProE1; P4, ProE1; P5, BasoE; P6, PolyE; P7, OrthoE; P8, Ortho/Ret). (C and D) Principal-components plots of (C) RNA-seq data and (D) ATAC-seq data, colored by FACS-sorted populations. Color code indicated below. (E) Number of chromatin accessibility peaks across populations. Error bars represent SEM number of peaks per population between replicates. (F) Heatmap showing relative numbers of differentially accessible peaks (top, blue shades) and differentially expressed genes (bottom, red shade) between populations.

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Experimental details: Tunable model of the human invasive TME enables multiplexed analysis of glioma markers and comparison to in vivo marker expression. a Diagram of brain TME model setup using patient-derived GSCs, human astrocytes, and human microglia in a hyaluronan-based matrix and 96-well tissue culture insert format. b Representative flow cytometry plots showing the ability to distinguish astrocyte, microglia, and glioma cell populations and determine glioma-specific proliferation (Ki67 + ), stemness (CD71 + ), and cell death. Isotype controls are shown in gray. Also shown is a representative fluorescence image of the porous membrane for invasion quantification. Scale bar is 50 um. c Representative fluorescence image within the gel with glioma cells (blue), astrocytes (green), and microglia (magenta). Scale bar is 50 um. d Quantification of cell viability under different media formulations for up to three days in hydrogel culture. e Quantification of cell viability following different enzymatic gel degradation protocols. f , g Comparison of %Ki67 + cells ( f ) and %CD71 + cells ( g ) for three in vitro cancer models and in vivo xenograft implants. In vitro data obtained by flow cytometry; in vivo data obtained from tissue sections, with the tumor border visually demarcated based on nuclear staining. Legends in f , g are the same. Comparisons conducted by unpaired t -tests, * p < 0.05 and ** p < 0.01 for n = 3-4.

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Experimental details: Fig. 1 T cell activation stimulates uptake and recycling of transferrin and TfR. A Transferrin uptake stimulation upon T cell activation. Jurkat T cells were equilibrated at 37 degC in the presence of transferrin-Alexa647 and subsequently left untouched (resting), or 30 min treated with non-stimulating solution (0 min activated) or 30 min stimulated with activating antibodies anti-CD3epsilon + anti-CD28. Uptake was stopped on ice and cells were analysed for transferrin-Alexa647 uptake. Displayed is the fold-change of Alexa647 signal relative to untouched (resting) controls. B Transiently boosted TfR recycling upon T cell activation. Jurkat T cells were incubated with biotinylated anti-TfR for 90 min to label all TfR pools with antibodies. Then, surface-exposed anti-TfR-biotin was blocked with unlabelled streptavidin. To assess TfR recycling upon activation, cells were activated with soluble activating antibodies (anti-CD3epsilon + anti-CD28) or left untreated for the indicated times. Antibody-labelled TfR delivered to the surface was detected with Pacific Blue-labelled streptavidin. Fold change of bound streptavidin-Pacific Blue in activated T cells is depicted relative to the corresponding resting cells. C Increased surface TfR levels upon T cell activation correlates with boosted TfR recycling. Jurkat T cells were activated with soluble activating antibodies (anti-CD3epsilon + anti-CD28) or left untreated for the indicated times and immediately fixed with 3.7% paraformaldeh

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Experimental details: TfR surface delivery and boosted recycling relies on flotillins. A Representative TIRF images of transferrin-Alexa488 (cyan) vesicle co-fusion events with mCherry Rab11a (magenta) in WT Jurkat T cells (top) or fusion with (middle) or without (bottom) mCherry-Rab11a in FlotKO Jurkat T cells. B Quantification of the percentage of transferrin-Alexa488 fusion events containing mCherry-Rab11a in WT and two FlotKO Jurkat T cell lines. Fusion events were counted as containing the indicated protein if a decrease in maximum intensity from the last three frames was greater than 10% compared to the maximum intensity of the initial frame. Data points represent means of independent experiments. C Quantification of TfR recycling boost in WT and FlotKO Jurkat T cells upon activation (left) and representative histograms (right) of n = 4 independent experiments. Cells were incubated with biotinylated anti-TfR for 90 min to label all TfR pools with antibodies. Then, surface-exposed anti-TfR-biotin was blocked with unlabelled streptavidin. To assess TfR recycling, cells were activated with soluble antibodies (anti-CD3epsilon + anti-CD28) or left untreated for the indicated times. Antibody-labelled TfR delivered to the surface was detected with Pacific Blue-labelled streptavidin. Fold change of bound streptavidin-Pacific Blue in activated T cells is depicted relative to resting cells at timepoint 0 min. D Increased surface TfR levels upon T cell activation in WT vs FlotKO Jurkat T cells. Quantif

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Experimental details: Fig. 2. Stage-dependent Ca 2+ influx into the EPCs upon stimulation with NMDA and glycine (GLY) in the absence or absence of MK-801. The size of cell population responding to the stimulation with NMDA/GLY (150 muM/50 muM) with Ca 2+ uptake was tested in the EPCs derived from nine different donors at days 8 , 12 , and 16 in culture. Changes in intracellular Ca 2+ were evaluated with Ca 2+ indicator FLUO-4. A : representative histograms showing fluorescence intensity in unstimulated cells (control, shown in green), cells stimulated with NMDA (150 muM) and glycine (50 muM) (NMDA/GLY, in blue), and stimulated cells in the presence (MK-801 + NMDA/GLY, in yellow) of antagonist MK-801 (60 muM). Blank (unstained) readouts from the cells free from fluorophore are shown in red. At day 8 of maturation one population of cells with high FLUO-4 intensity (gate A) and one with low FLUO-4 intensity (gate B) were present. At later stages only one population was present (gate A). Quantification of the data is presented in Table 2 . B : dot plots illustrating relative change of intracellular Ca 2+ levels in CD71 + cells (gate A +- and gate A ++ ). Presented are the representative readouts from the EPCs at day 12 in culture in which Ca 2+ -sensitive fluorescence of FLUO-4 was recorded in unstimulated cells (control), in stimulated (NMDA/GLY) cells, and in stimulated cells in the presence of antagonist (MK-801 + NMDA/GLY). Quantification of the data is presented in Table 3 . C : Ca 2+ uptake by t

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Experimental details: Extended Figure 2 Human CD34 + Erythroid Differentiation System a , Total CFU-E colonies formed during day 0-9. CFU-E colony numbers were quantified by plating 1000 cells from various time points during day 0-9 of the human CD34 + erythroid culture on methylcellulose. CFU-E colonies were quantified after 12-14 days. Total CFU-E colony numbers in culture under conditions without GW7647 (black line) or with GW7647 (red line) were calculated using the total cell numbers at corresponding time points in Figure 2a. b , Human CD34+ cells were treated at day 1 with 100 nM GW7647 with or without DEX at the concentration indicated in the figure. At day 6, total cell numbers were counted and cells were collected for BFU-E colony assays. c , Protein expression of PPARalpha demonstrating shRNA knock-down efficiency via lentiviral transduction. LacZ shRNA is used as a control. shRNA-1 and -2 are both specific for PPARalpha . shRNA-2 has higher knock-down efficiency. d , Cell pellets of 1 million cells demonstrating hemoglobin accumulation during the differentiation process. e , Flow cytometry analyses of erythroid markers during the 21-day human CD34 + erythroid culture. (top row) c-kit vs. CD235a; (middle row) CD71 vs. CD235a. Note the sequential induction of c-kit, CD71 and CD235a, as well as the sequential down-regulation of c-kit and CD71, (bottom row) Enucleated reticulocytes are CD235a + Hoechst - , nuclei are CD235a - Hoechst + , and nucleated erythroblasts are CD235a + Hoechst + .

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Experimental details: Extended Figure 3 GW7647 increases erythroid progenitors and CD235a + cells in RPS19 knock down human progenitor cells a , Human CD34 + hematopoietic progenitors were transduced with lentivirus encoding GFP and either a scrambled shRNA or an shRNA targeting RPS19. Then transduced cells were treated with or without 100 nM GW7647. After 48 hrs, GFP + cells were sorted by FACS and plated for BFU-E and CFU-E colony forming assays. RPS19 knocking down efficiency is shown at the bottom. (* p < 0.05; Student t test. Error bars represent mean +- S.D. from three independent experiments.) b. Sorted GFP + cells were returned to culture with the indicated concentration of GW7647. (Top) Percentage of CD71 + cells at day 9 in was determined by FACS; (Bottom) Percentage of CD235a + cells at day 21 was determined by FACS. (* p < 0.05; Student t test. Error bars represent mean +- S.D. from three independent experiments.) c. Total cell numbers generated from one GFP positive cell at the indicated times of culture. (* p < 0.05; Student t test. Error bars represent mean +- S.D. from three independent experiments.)

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Experimental details: Figure 1--figure supplement 3. Pooled shRNA screen in primary HSPCs undergoing erythroid differentiation. ( A ) Histogram showing distribution of number of independent hairpins included in the library to target each of the candidate's genes. ( B ) Representative FACS plots of erythroid cell surface markers CD71 (transferrin receptor) and CD235a (Glycophorin A) expression at various time points during erythroid differentiation at which deep sequencing of shRNAs was performed. Percentages in each quadrant is represented as mean and standard deviation of 3 experiments from independent donors that were uninfected (Mock) or infected with the shRNA library (Pool).

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Experimental details: Figure 6. SF3A2 is a Key regulator of Human Erythropoiesis and Modulates Erythropoiesis Defects in a Murine Model of MDS. ( A ) Quantitative RT-PCR and ( B ) Western blot showing the expression of SF3A2 in human CD34 + cells five days post-infection with the respective lentiviral shRNAs targeting SF3A2 (sh1-4) and a control luciferase gene (shLUC). ( C ) Growth curves showing that downregulation of SF3A2 results in reduced total cell numbers during erythroid differentiation from three independent experiments. ( D ) Representative FACS plots of erythroid cell surface markers CD71 (transferrin receptor) and CD235a (Glycophorin A) expression at various time points during erythroid differentiation. Percentages in each quadrant are represented as mean and standard deviation of three independent experiments ( E ) Altered splicing events identified by RNA-Seq analysis of stage matched erythroid cells (shSF3A2 vs. shLUC). Overlapping changes observed in SF3B1 mutant BM cells from MDS patients (Obeng et al) ( Figure 6--source data 5 and 6 ). Differentially expressed genes and pathway analysis available in Figure 6--source data 1 - 4 . ( F ) Lineage negative bone marrow cells from wildtype (WT) and Sf3b1 K700E mice were infected with shRNAs targeting murine Sf3a2 gene co-expressing a reporter GFP gene. Percentage of Ter119 + CD71 + erythroid cells within the GFP compartment after 48 hr in erythroid differentiation. ( G ) Total cell numbers of GFP + erythroid cells after