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	48-602MAG	Buffer Detection Kit for Magnetic Beads	1 Kit

17-10146 Sigma-AldrichLentiBrite™ Calreticulin-RFP-KDEL Lentiviral Biosensor

MSDS (material safety data sheet) or SDS, CoA and CoQ, dossiers, brochures and other available documents.

SDS
CoA
Technical Information

Key Applications: Transfection, Immunofluorescence, Immunocytochemistry

Recommended Products

Přehled

Replacement Information

Tabulka spec. kláve

Key Applications	Detection Methods
TFX, IF, ICC	Fluorescent

Description
Catalogue Number	17-10146
Trade Name	LentiBrite Chemicon
Description	LentiBrite™ Calreticulin-RFP-KDEL Lentiviral Biosensor
Overview	*Read our application note in Nature Methods!* http://www.nature.com/app_notes/nmeth/2012/121007/pdf/an8620.pdf (Click Here!) Learn more about the advantages of our LentiBrite Lentiviral Biosensors! Click Here Biosensors can be used to detect the presence/absence of a particular protein as well as the subcellular location of that protein within the live state of a cell. Fluorescent tags are often desired as a means to visualize the protein of interest within a cell by either fluorescent microscopy or time-lapse video capture. Visualizing live cells without disruption allows researchers to observe cellular conditions in real time. Lentiviral vector systems are a popular research tool used to introduce gene products into cells. Lentiviral transfection has advantages over non-viral methods such as chemical-based transfection including higher-efficiency transfection of dividing and non-dividing cells, long-term stable expression of the transgene, and low immunogenicity. EMD Millipore is introducing LentiBrite™ Lentiviral Biosensors, a new suite of pre-packaged lentiviral particles encoding important and foundational proteins of autophagy, apoptosis, and cell structure for visualization under different cell/disease states in live cell and in vitro analysis. Pre-packaged, fluorescently-tagged with GFP & RFP Higher efficiency transfection as compared to traditional chemical-based and other non-viral-based transfection methods Ability to transfect dividing, non-dividing, and difficult-to-transfect cell types, such as primary cells or stem cells Non-disruptive towards cellular function EMD Millipore’s LentiBrite™ Calreticulin-RFP-KDEL lentiviral particles provide bright fluorescence and precise localization to enable live cell analysis of autophagy in difficult-to-transfect cell types.
Alternate Names	Endoplasmic reticulum resident protein 60 Calregulin
Background Information	Calreticulin is a resident protein of the lumen of the endoplasmic reticulum that plays important roles in protein folding and calcium homeostasis. In addition, cells induced to undergo apoptosis with selected agents allow passage of calreticulin from the ER to the cell surface, and the cell surface-exposed calreticulin promotes engulfment by phagocytic cells, a process termed immunogenic cell death. Expression of genetic fusions between calreticulin and fluorescent proteins has permitted analysis of the dynamics of chaperone-substrate interactions and immunogenic cell death. EMD Millipore’s LentiBrite™ Calreticulin-RFP-KDEL lentiviral particles provide bright fluorescence and precise localization to enable live cell analysis of calreticulin dynamics in difficult-to-transfect cell types.

References

Product Information
Components	Calreticulin-TagRFP-KDEL Lentivirus : One vial containing 25 µL of lentiviral particles at a minimum of 3 x 10E8 infectious units (IFU) per mL. For lot-specific titer information, please see “Viral Titer” in the product box above. Promoter EF-1 (Elongation Factor-1) Multiplicty of Infection (MOI) MOI = Ratio of # of infectious lentiviral particles (IFU) to # of cells being infected. Typical MOI values for high transduction efficiency and signal intensity are in the range of 20-40. For this target, some cell types may require lower MOIs (e.g., HT-1080, HeLa, U2OS), while others may require higher MOIs (e.g., human mesenchymal stem cells (HuMSC), human umbilical vein endothelial cells (HUVEC)). NOTE: MOI should be titrated and optimized by the end user for each cell type and lentiviral target to achieve desired transduction efficiency and signal intensity.
Detection method	Fluorescent
Quality Level	MQ100

Applications
Key Applications	Transfection Immunofluorescence Immunocytochemistry
Application Notes	Fluorescence Microscopy Imaging: (See Figure 1 in datasheet) U2OS cells were plated in a chamber slide and transduced with lentiviral particles at an MOI of 5 for 24 hours. After media replacement and 48 hours further incubation, cells were fixed with formaldehyde and mounted. Images were obtained by oil immersion wide-field fluorescence microscopy. The calreticulin-RFP-KDEL appears in a meshwork pattern in the cytoplasm. Immunocytochemistry Comparison and Inhibitor Analysis: (See Figure 2 in datasheet) Similar to Figure 1, HeLa cells were plated in a chamber slide and transduced with lentiviral particles at an MOI of 40 for 24 hours. After 24 hours, media was replaced and cells were then further incubated for 48 hours. Immunocytochemical staining (green) of the same fields of view with a polyclonal antibody against calreticulin reveals similar expression patterns to the RFP-protein (red). Hard-to-transfect Cell Types: (See Figure 3 in datasheet) Primary cell types HUVEC and HuMSC were plated in chamber slides and transduced with lentiviral particles at an MOI of 40 and 10 respectively for 24 hours. Additional Cell Type: (See Figure 4 in datasheet) HT1080 and REF52 cells were treated as in Figure 1. For optimal fluorescent visualization, it is recommended to analyze the target expression level within 24-48 hrs after transfection/infection for optimal live cell analysis, as fluorescent intensity may dim over time, especially in difficult-to-transfect cell lines. Infected cells may be frozen down after successful transfection/infection and thawed in culture to retain positive fluorescent expression beyond 24-48 hrs. Length and intensity of fluorescent expression varies between cell lines. Higher MOIs may be required for difficult-to-transfect cell lines.

Applications

Key Applications

Transfection
Immunofluorescence
Immunocytochemistry

Application Notes

Fluorescence Microscopy Imaging:
(See Figure 1 in datasheet)
U2OS cells were plated in a chamber slide and transduced with lentiviral particles at an MOI of 5 for 24 hours. After media replacement and 48 hours further incubation, cells were fixed with formaldehyde and mounted. Images were obtained by oil immersion wide-field fluorescence microscopy.
The calreticulin-RFP-KDEL appears in a meshwork pattern in the cytoplasm.

Immunocytochemistry Comparison and Inhibitor Analysis:
(See Figure 2 in datasheet)
Similar to Figure 1, HeLa cells were plated in a chamber slide and transduced with lentiviral particles at an MOI of 40 for 24 hours. After 24 hours, media was replaced and cells were then further incubated for 48 hours. Immunocytochemical staining (green) of the same fields of view with a polyclonal antibody against calreticulin reveals similar expression patterns to the RFP-protein (red).

Hard-to-transfect Cell Types:
(See Figure 3 in datasheet)
Primary cell types HUVEC and HuMSC were plated in chamber slides and transduced with lentiviral particles at an MOI of 40 and 10 respectively for 24 hours.

Additional Cell Type:
(See Figure 4 in datasheet)
HT1080 and REF52 cells were treated as in Figure 1.

For optimal fluorescent visualization, it is recommended to analyze the target expression level within 24-48 hrs after transfection/infection for optimal live cell analysis, as fluorescent intensity may dim over time, especially in difficult-to-transfect cell lines. Infected cells may be frozen down after successful transfection/infection and thawed in culture to retain positive fluorescent expression beyond 24-48 hrs. Length and intensity of fluorescent expression varies between cell lines. Higher MOIs may be required for difficult-to-transfect cell lines.

Biological Information
Gene Symbol	CRP55 ERp60 HACBP Grp60 CALR CRTC
Purification Method	PEG precipitation
UniProt Number	P27797

Physicochemical Information

Dimensions

Materials Information

Toxicological Information

Safety Information according to GHS

Safety Information

Product Usage Statements
Quality Assurance	Evaluated by transduction of HT-1080 cells and fluorescent imaging performed for assessment of target localization and transduction efficiency.
Usage Statement	This product contains genetically modified organisms (GMO). Within the EU GMOs are regulated by Directives 2001/18/EC and 2009/41/EC of the European Parliament and of the Council and their national implementation in the member States respectively. This legislation obliges Merck to request certain information about you and the establishment where the GMOs are being handled. Click here for Enduser Declaration (EUD) Form. Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.

Product Usage Statements

Quality Assurance

Evaluated by transduction of HT-1080 cells and fluorescent imaging performed for assessment of target localization and transduction efficiency.

Usage Statement

This product contains genetically modified organisms (GMO). Within the EU GMOs are regulated by Directives 2001/18/EC and 2009/41/EC of the European Parliament and of the Council and their national implementation in the member States respectively. This legislation obliges Merck to request certain information about you and the establishment where the GMOs are being handled. Click here for Enduser Declaration (EUD) Form.
Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.

Storage and Shipping Information
Storage Conditions	Storage and Handling Lentivirus is stable for at least 4 months from date of receipt when stored at -80°C. After first thaw, place immediately on ice and freeze in working aliquots at -80°C. Frozen aliquots may be stored for at least 2 months. Further freeze/thaws may result in decreased virus titer and transduction efficiency. IMPORTANT SAFETY NOTE Replication-defective lentiviral vectors, such as the 3rd Generation vector provided in this product, are not known to cause any diseases in humans or animals. However, lentiviruses can integrate into the host cell genome and thus pose some risk of insertional mutagenesis. Material is a Risk Group 2 and should be handled under BSL2 controls. A detailed discussion of biosafety of lentiviral vectors is provided in Pauwels, K. et al. (2009). State-of-the-art lentiviral vectors for research use: Risk assessment and biosafety recommendations. Curr. Gene Ther. 9: 459-474.

Storage and Shipping Information

Storage Conditions

Storage and Handling
Lentivirus is stable for at least 4 months from date of receipt when stored at -80°C. After first thaw, place immediately on ice and freeze in working aliquots at -80°C. Frozen aliquots may be stored for at least 2 months. Further freeze/thaws may result in decreased virus titer and transduction efficiency.

IMPORTANT SAFETY NOTE
Replication-defective lentiviral vectors, such as the 3rd Generation vector provided in this product, are not known to cause any diseases in humans or animals. However, lentiviruses can integrate into the host cell genome and thus pose some risk of insertional mutagenesis. Material is a Risk Group 2 and should be handled under BSL2 controls. A detailed discussion of biosafety of lentiviral vectors is provided in Pauwels, K. et al. (2009). State-of-the-art lentiviral vectors for research use: Risk assessment and biosafety recommendations. Curr. Gene Ther. 9: 459-474.

Packaging Information
Material Size	1 vial (minimum of 3 x 10E8 IFU/mL)

Transport Information

Supplemental Information

Specifications

Global Trade Item Number
Katalogové číslo	GTIN
17-10146	04053252639999

Documentation

LentiBrite™ Calreticulin-RFP-KDEL Lentiviral Biosensor MSDS

Title
Safety Data Sheet (SDS)

LentiBrite™ Calreticulin-RFP-KDEL Lentiviral Biosensor Certificates of Analysis

Title	Lot Number
LentiBrite Calreticulin-RFP-KDEL Lentiviral Biosensor - N1956534	N1956534
LentiBrite Calreticulin-RFP-KDEL -2763837	2763837
LentiBrite Calreticulin-RFP-KDEL Lentiviral Biosensor - 3321010	3321010
LentiBrite Calreticulin-RFP-KDEL Lentiviral Biosensor - 3598986	3598986
LentiBrite Calreticulin-RFP-KDEL Lentiviral Biosensor - 3722047	3722047
LentiBrite Calreticulin-RFP-KDEL Lentiviral Biosensor - 3989489	3989489
LentiBrite Calreticulin-RFP-KDEL Lentiviral Biosensor - 4028549	4028549
LentiBrite Calreticulin-RFP-KDEL Lentiviral Biosensor Packaged Lentiviral Particles	3134713
LentiBrite™ Calreticulin-RFP-KDEL Lentiviral Biosensor - 2458010	2458010
LentiBrite™ Calreticulin-RFP-KDEL Lentiviral Biosensor - 2282198	2282198

Technical Info

Title
LentiBrite™ Lentiviral Biosensors for Fluorescent Cellular Imaging: Analysis of Autophagosome Formation

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Kategorie

Life Science Research > Cell Analysis > Cell-based Assays > Lentiviral Biosensors

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