Immunofluorescence Analysis of GLUT1 Expression

Background and Purpose: 

Glucose is the primary energy source for almost all living cells and glucose uptake from the blood stream into the cells is critical for the survival of cells. Due to the polar nature of glucose molecule, it cannot traverse the plasma membrane and therefore, needs transporters for entry into the cells. There are two distinct type of glucose transporters, sodium dependent glucose linked transporters (SGLTs) and facilitative glucose transporters (GLUTs). SGLTs are primarily expressed in intestinal lining and renal tubules where they absorb glucose against the gradient in an ATP dependent manner. Gluts are facilitative transporters and they facilitate glucose uptake in to the cells along the gradient. GLUT proteins contain 12 transmembrane membrane domains with intracellular amino and carboxyl terminals. Based on multiple sequence alignment studies, GLUTs have been subdivided into three distinct subclasses (Class I, II and II). Class I comprises of four proteins, GLUT1, GLUT2, GLUT3 and GLUT4. GLUT2 is mainly expressed in pancreatic β-cells, liver and kidney. GLUT2 acts as a glucose sensor in liver where it facilities both entry and release of glucose from hepatocytes. GLUT3 is a high affinity glucose transporter and is predominantly expressed in brain where the requirement of glucose is high. GLUT4 is an insulin-dependent transporter and is expressed in insulin sensitive cells like adipose and skeletal muscle. In fasting condition, GLUT4 is predominantly localized in intracellular spaces and insulin release in the postprandial state triggers the translocation of GLUT4 to the cell surface where it precipitates in glucose transport activity.   GLUT1 is expressed in almost all type of cells, in addition, its expression is high in rapidly proliferating cells. For example, erythrocytes, which rely solely on non-oxidative glucose metabolism, have very high GLUT1 expression. GLUT1 has been comprehensively studied in health and disease and abnormal expression of GLUT1 is found in a wide spectrum of epithelial malignancies. Cancer cells need high energy levels via glycolytic generation of ATP to proliferate and survive (1). GLUT1 overexpression in cancer cells is one of the primary mechanisms by which malignant cells sustain growth and proliferation at the expense of host cells. Several independent studies have demonstrated that the overexpression of GLUT1 is associated with poor survival in most solid tumors, signifying that the expression status of GLUT1 is an important prognostic indicator and a promising therapeutic target (2-4). Therefore, accurate assessment of GLUT1 expression in cells is important. Here we describe an immunofluorescence-based quantification of GLUT1 expression using single cell analysis. 
 

Materials and Reagents:

1. 1.5 ml Eppendorf tubes.
2. Glass coverslips.
3. 1X Phosphate buffered saline (PBS).
4. Formaldehyde solution, 36.5-38% (Sigma-Aldrich, catalog number: F8775).
5. Triton X-100 (Sigma-Aldrich, catalog number: X100).
6. Fetal Bovine serum (FBS) from Glibco (catalogue number: A3840001).
7. 37 °C incubator.
8. Anti-glut1 antibody (Thermo Fisher Scientific, Catalogue #PA1-46152).
9. Fluorescein isothiocyanate Alexa Flour 488 conjugated antibody (Cat # A-11034).
    

Equipment:

MetaXpress High-Content Image Acquisition system (Molecular Devices)
 

Software:

1. Metamorph image processing software (Molecular devices).
2. Microsoft Excel.
3. GraphPad Prism.
    

Procedure:

Cells used in this protocol were primary dermal fibroblasts isolated from patients using explant method. In addition, Hs27 cells (normal fibroblast) were also used after genetic modification using CRISPR.

Preparation of cells: 

1. Prepare 4% Paraformaldehyde in 1X PBS (Refer to Recipe section).  
2. Grow 5 x 10³ – 10 x 10³ cells on each well of a clear-bottom 96 well plate ( Corning, Cat # 3904)
3. Incubate plates with the cells in the CO2 incubator at 37 °C overnight.
4. Wash cells with PBS. 
5. Fix cells by incubating the cells with 30-40 μl of 4% formaldehyde solution for 15 minutes at room temperature.
6. Wash cells three times with PBS. 
7. Incubate cells with (1:1000) anti-GLUT1 antibody in antibody incubation buffer for 30 minutes at 37 °C.
8. Wash cells four times with PBS.
9. Incubate cells with (1:1000) fluorescein isothiocyanate Alexa Flour 488 conjugated antibody in antibody incubation buffer for 30 min.
10. Wash cells four times with PBS.
11. Image cells immediately on MetaXpress High-Content Image Acquisition system using GFP/FITC filters.

Analysis of GLUT1 expression:

1. Image analysis was carried out to determine fluorescent intensity of GLUT1 using image processing software Metamorph (Molecular Devices). Fluorescent intensity was determined for each cell, which and an average of at least 100 cells was used to quantify the expression of GLUT1. The method has been described before to quantify β2-Adrenergic receptor (5)
2. Analyse data on excel to generate a figure of the evaluation of GLUT1 activity in these cells.
    

Recipes:

1. 4% Paraformaldehyde in 1X PBS
   150 ul of Formaldehyde solution, 36.5-38%
   1350 ul of 1X PBS
2. Antibody incubation buffer- (5% Fetal Bovine Serum and 0.05% Triton in PBS).
   5 ul FBS
   0.5 ul Trition X-100
   994.5 ul PBS
    

References 

1.  Oh S, Kim H, Nam K, Shin I. Glut1 promotes cell proliferation, migration and invasion by regulating epidermal growth factor receptor and integrin signaling in triple-negative breast cancer cells. BMB Rep. 2017;50(3):132-7.

2. Shim BY, Jung JH, Lee KM, Kim HJ, Hong SH, Kim SH, et al. Glucose transporter 1 (GLUT1) of anaerobic glycolysis as predictive and prognostic values in neoadjuvant chemoradiotherapy and laparoscopic surgery for locally advanced rectal cancer. Int J Colorectal Dis. 2013;28(3):375-83.

3.  Shen YM, Arbman G, Olsson B, Sun XF. Overexpression of GLUT1 in colorectal cancer is independently associated with poor prognosis. Int J Biol Markers. 2011;26(3):166-72.

4.  Saigusa S, Toiyama Y, Tanaka K, Okugawa Y, Fujikawa H, Matsushita K, et al. Prognostic significance of glucose transporter-1 (GLUT1) gene expression in rectal cancer after preoperative chemoradiotherapy. Surg Today. 2012;42(5):460-9.

5.  Haji E, Mahri SA, Aloraij Y, Malik S, Mohammad S. Single-cell Analysis of beta2-Adrenergic Receptor Dynamics by Quantitative Fluorescence Microscopy. Curr Mol Med. 2020;20(6):488-93.

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