Animal Culture Protocol PDF
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This document contains protocols for animal cell culture, including aseptic technique, subculturing, cell quantification, and cryopreservation. These methods describe the fundamental practices for maintaining and growing cells for research or other purposes.
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**Protocol 1: Aseptic Technique and Good Cell Culture Practice** 1\. **Why is it important to sanitise the microbiological safety cabinet before starting work?** Sanitising the cabinet prevents contamination from bacteria, fungi, and other sources, ensuring the cell culture environment remains s...
**Protocol 1: Aseptic Technique and Good Cell Culture Practice** 1\. **Why is it important to sanitise the microbiological safety cabinet before starting work?** Sanitising the cabinet prevents contamination from bacteria, fungi, and other sources, ensuring the cell culture environment remains sterile. 2\. **What is the purpose of using 70% alcohol in aseptic techniques?** 70% alcohol effectively disinfects surfaces and gloves by killing microorganisms without evaporating too quickly, which allows sufficient contact time for disinfection. 3\. **Describe the steps involved in sanitising gloves before handling cell culture materials.** Spray gloves with 70% alcohol and let them air dry for 30 seconds before beginning work. 4\. **What precautions should be taken to avoid contamination while working in the cabinet?** Avoid touching the face or hair, move slowly, avoid rapid movements, and direct speech, sneezes, or coughs away from the cabinet. 5\. **Why is it necessary to move slowly inside and around the cabinet?** Slow movements allow proper airflow circulation within the cabinet, preventing disruption of the sterile environment. 6\. **How should liquid cell culture waste be handled and discarded?** Liquid waste should be discarded into sodium hypochlorite (10,000 ppm) and kept in the cabinet for at least two hours (preferably overnight) before discarding into the drain with copious amounts of water. 7\. **Explain the importance of periodic cleaning or fumigating the cabinet.** Periodic cleaning or fumigation ensures long-term sterility by eliminating contaminants that may accumulate over time. 8\. **What is the recommended procedure if gloves become contaminated during cell culture work?** Respray the gloves with 70% alcohol and allow them to air dry before continuing work. **Protocol 2: Subculture of Adherent Cell Lines** 1\. **What is the purpose of subculturing adherent cell lines?** Subculturing prevents cell death by providing more space and nutrients when cells reach confluency or the medium is depleted. 2\. **Why is trypsin/EDTA used in the subculturing process, and when should it not be used?** Trypsin/EDTA detaches cells by breaking down proteins that mediate cell adhesion. It should not be used for cells sensitive to proteases or when enzymatic detachment removes membrane markers or receptors of interest. 3\. **List the materials and equipment required for subculturing adherent cells.** Materials: Pre-warmed medium, PBS without Ca²⁺/Mg²⁺, trypsin/EDTA, trypsin inhibitor (if necessary), and sterile tissue. Equipment: PPE, microbiological safety cabinet, water bath, inverted microscope, centrifuge, pre-labelled flasks, haemocytometer or cell counter, pipettes, and marker pen. 4\. **Describe the procedure for detaching cells from the flask using trypsin.** Remove spent medium, wash the cell monolayer with PBS, add trypsin/EDTA, rotate the flask to coat the monolayer, decant excess trypsin, and incubate for 2--10 minutes until cells detach. 5\. **How is trypsin activity neutralised after cell detachment?** Add a serum-containing medium to the detached cells to inactivate trypsin. For serum-free cultures, use a trypsin inhibitor like soybean trypsin inhibitor. 6\. **Why is it important to confirm the absence of contaminants before starting subculture?** Contaminants can compromise cell growth and experiment reliability, making it essential to start with a clean culture. 7\. **What is the role of the inverted microscope in the subculture process?** The inverted microscope is used to assess cell confluency, confirm detachment, and check for contaminants. 8\. **How do you determine the correct seeding density for transferring cells to a new flask?** Refer to the ECACC cell line data sheet for the specific seeding density and transfer the required number of cells accordingly. 9\. **What are the steps to perform a cell count after subculturing?** Take 100--200 μL of the cell suspension, mix with trypan blue, and count cells using a haemocytometer or automated cell counter. 10\. **What alternative methods can be used for detaching cells if proteases are not suitable?** Cells can be mechanically detached using cell scrapers in a small volume of medium. **Protocol 3: Cell Quantification** 1\. **Why is it important to quantify cells before manipulations like transfections or cryopreservation?** Quantifying cells ensures a consistent cell number, which maintains optimal growth conditions and standardises procedures, leading to reproducible results. 2\. **What is the purpose of Trypan Blue in cell quantification?** Trypan Blue is a vital stain used to differentiate viable cells (bright, unstained) from non-viable cells (stained blue). 3\. **How do you prepare a haemocytometer for cell counting?** Clean the haemocytometer, moisten the coverslip with water or exhaled breath, and slide it over the chamber until Newton's refraction rings appear. 4\. **What is the significance of Newton's refraction rings during haemocytometer preparation?** These rings indicate proper placement of the coverslip, ensuring accurate cell counting. 5\. **Describe the procedure for loading a haemocytometer with a cell suspension.** Fill both sides of the chamber with 5--10 μL of cell suspension and view under an inverted microscope at x20 magnification. 6\. **How do you calculate cell concentration from a haemocytometer?** Count viable and non-viable cells in multiple squares, calculate the concentration using the haemocytometer's specific formula, and determine the percentage of viable cells. 7\. **Why is it recommended to count \>100 cells when performing cell quantification?** Counting more cells increases the accuracy and reliability of the cell concentration and viability data. **Protocol 4: Cryopreservation of Cell Lines** 1\. **What is the aim of cryopreserving cell lines?** Cryopreservation allows long-term storage of cell lines, maintaining their viability and characteristics for future use. 2\. **When is the best time to harvest adherent cells for cryopreservation?** Cells should be harvested in the log phase of growth, ideally at 80--90% confluency. 3\. **What is the purpose of freeze medium, and what does it typically contain?** Freeze medium protects cells from damage during freezing. It commonly contains 90% fetal bovine serum (FBS) and 10% dimethyl sulfoxide (DMSO) or glycerol. 4\. **Why is it important to achieve \>90% cell viability before freezing?** High cell viability ensures better recovery and growth after thawing. 5\. **Describe the process of resuspending cells for cryopreservation.** After centrifugation, resuspend cells at a concentration of 2--4 x 10⁶ cells/mL in freeze medium. 6\. **What information should be labelled on cryopreservation ampoules?** Ampoules should include the cell line name, passage number, lot number, cell concentration, and the date. 7\. **What is the purpose of using a passive freezer like the Nalgene Mr Frosty box?** The passive freezer ensures a controlled cooling rate of approximately -1°C per minute, which is optimal for preserving cell viability during freezing. 8\. **Where should frozen ampoules be stored after initial freezing at -80°C?** Ampoules should be transferred to the vapor phase of a liquid nitrogen storage vessel for long-term preservation. 9\. **Why is it necessary to record the storage locations of cryopreserved ampoules?** Proper records ensure easy retrieval and prevent loss or misidentification of samples.