Protocol CloneMedia-CHO Semi-solid media for the growth of CHO-S Colonies Control #: 03PCT1010.A0 Effective Date: 01-Jul-11 ECO #: 4002
Content Page Introduction 3 Protocol 4 1. Media Preparation 4 2. Protocol for plating of CHO-S (Serum-free, suspension adapted CHO) 5 3. Protocol for plating of adherent CHO (In the presence of serum) 7 3.1. Plating adherent CHO as suspended colonies in semi-solid media 8 3.2. Plating of CHO as adherent colonies 9 Storage Store CloneMedia-CHO at -20 C Prior to use thaw CloneMedia-CHO at 2 C 8 C overnight. Do not shake contents until completely defrosted. Allow to adjust to room temperature prior to using. Once defrosted, CloneMedia-CHO may be stored at 2 C 8 C for up to one week. For Research Use Only Molecular Devices > 2 of 10
Introduction CloneMedia-CHO is a complete semi-solid media designed for the growth of colonies of CHO cells (CHO-S or adherent CHO). Developed specifically for optimised use with Molecular Devices ClonePix mammalian colony picking systems, CloneMedia-CHO supports colony formation as well as the fluorescent assay of secreted product using CloneDetect Agent. Supplied as 90ml in 100ml bottles, supplements and selective agents can be added for optimal growth of your CHO cell line without the need to aliquot. CloneMedia-CHO is free of animal sourced products and is suitable for users wishing to work under chemically-defined conditions. CloneMedia-CHO G (K8740) is the glutamine free equivalent of CloneMedia-CHO (K8710). Molecular Devices > 3 of 10
Protocol Please note: The recommended plating procedures differ for serum-free, suspension-adapted CHO lines and adherent CHO lines grown in the presence of serum. Follow general instructions for Media Preparation (Part 1) first and then select and follow the appropriate instructions for Cell Plating (Part 2.1 for plating of CHO-S and Part 2.2 for plating of adherent CHO). 1. Media Preparation Semi-solid media prepared from 90ml of CloneMedia-CHO is sufficient for the following; Up to ten Molecular Devices PetriWell-1 Plates or 100mm tissue culture dishes (plate 9ml of media each) Or Up to eight PetriWell-6 Plates (plate 2ml of media per well). Prior to use, thaw CloneMedia-CHO at 2 C 8 C overnight. Do not shake contents until completely defrosted. Allow to adjust to room temperature prior to use (do not pre-warm to 37 C). Prepare media from components adjusted to room temperature, only. Defrosted CloneMedia-CHO should be used as soon as possible, but can be stored at 2 C 8 C for up to one week. For 100ml of semi-solid media add any additional components necessary (e.g. selection agents, antibiotics) to the bottle containing 90ml of CloneMedia-CHO. Bring up to a final volume of 100ml with sterile tissue culture water if necessary. Should the volume of additional components exceed 10ml, ensure that the final volume of the media does not exceed 110ml. Shake bottle vigorously for ca. 30 seconds to mix thoroughly. Allow time for bubbles to escape (ca. 10 minutes at room temperature). Any small bubbles remaining will disperse in the plate during the culture period. For fluorescent detection assays, CloneDetect Agent may be added at this stage (see list of CloneDetect Agent products). Mix CloneDetect gently into the prepared media and protect bottle from direct light (please refer to the CloneDetect protocol for detailed instructions). Please select and follow plating instructions applicable for your CHO cell type from the sections below. Molecular Devices > 4 of 10
2. Plating of CHO-S (Serum-free, suspensionadapted CHO) It is recommended that conditions for successful plating are established with a stable transfected cell line before applying this to a recently transfected population where the window for selection of colonies is small. Cell Lines can also be plated for analysis of the stability of the population or for recloning. Suspension-adapted CHO cells growing in semi-solid conditions form discreet, spherical colonies suspended in the semi-solid media. (See Figure 1). Figure 1: Images of colonies of CHO-S cells grown in serum-free, semi-solid media taken on day 8 post-plating. Images were taken using Molecular Devices CloneSelect Imager. The correct density of colonies in the culture dish is key to the success of colony selection and the automated picking process. It is therefore crucial to thoroughly optimise the seeding densities. Seeding densities required to achieve an optimal density of colonies depend on the inherent plating efficiency of the cells used as well as the viability and growth phase of the cell suspension culture at the time of plating. The following ranges of seeding densities may serve as a guide line for your optimisation; Stable and robust CHO-S cell lines (e.g. parental CHO-S cell line): 250 cells/ml, 500 cells/ml, 1000 cells/ml) Other CHO-S lines (e.g. non-clonal expressing cell line): 500 cells/ml, 1000 cells/ml, 2000 cells/ml Fresh transfections: Molecular Devices > 5 of 10
1x103 cells/ml, 2x103 cells/ml, 1x102-8 cells/ml Please note: When plating fresh transfections the optimal seeding density is highly dependent on the transfection efficiency as well as the kinetics and efficiency of selection. Cells may require a period of recovery prior to seeding in semi-solid media. The selection pressure applied may also require optimisation. Cells should be plated 48 hours after their last passage and should have viability greater than 95%. Determine the volume of culture to be added for optimal seeding density (e.g. by haemocytometer). Add correct amount of cells to the semi-solid media and invert several times to gently to ensure even distribution. To ensure clonality it may be necessary to dissociate cells by gently pipetting up and down the cell suspension but care must be taken not to damage the cells. Dispense 2ml per well of a Molecular Devices PetriWell-6 Plate or 9ml of media per Molecular Devices PetriWell-1 Plate or 100mm tissue culture dish. This may be achieved using a 10ml pipette or by pouring. Tilt the culture dish gently to ensure even distribution. The incubation time required for CHO-S in semi-solid media requires precautions to keep the semi-solid media well hydrated. To this end fill areas of the plate between wells with ca. 2-4ml of sterile water. Place cultures in an incubator at 37 C, 5% CO2 for 7-14 days to allow colonies to grow. Verify the presence of growing colonies at a suitable time point using a light microscope. Do not disturb plates from the incubator before day 4. At that time, small colonies of 25-100 cells should be visible. Avoid repeated checking of the plates. If necessary, have a specific imaging plate which will not form part of the main batch. This can be used to confirm the progress of colony growth and determine an appropriate window for picking. Molecular Devices > 6 of 10
3.0 Plating of adherent CHO (in the presence of serum) For adherent CHO lines, two different plating protocols have been developed for the growth of colonies in semi-solid media; 3.1. CHO-plating for suspended colonies 3.2. CHO-plating for adherent colonies Molecular Devices ClonePix systems are capable of picking suspended colonies as well as adherent colonies growing at the bottom of the culture dish providing that they are discreet from one another (see Figure 2 below for typical images). Protocol 3.1 is the preferred method as suspended colonies offer the inherent advantage of being more compact, which allows clones to be plated and picked at a higher density with low risk of cross contamination. However, not all adherent CHO lines are suitable for this approach. Depending on the properties of the CHO line used, cells will more or less readily adhere to the bottom of the culture dish and form a layer of adherent cells in the background. For highly adherent CHO lines, protocol 3.2 will be more suitable. Suspended colonies Adherent colonies Figure 2: Images of CHO colonies in semi-solid media prepared using CloneMatrix. Cells grow as suspended colonies (left panel) when plated according to protocol 3.1. Adherent colonies (right panel) obtained by plating according to protocol 3.2. Images were taken using a white light microscope at 2-80X magnification on day 10 post-plating. Molecular Devices > 7 of 10
3.1 CHO-plating for suspended colonies Prepare semi-solid media as described above and supplement with a small amount of serum (1% or less if possible). The correct density of colonies in the culture dish is key to the success of colony selection and the automated picking process. It is therefore crucial to thoroughly optimise the seeding densities. Seeding densities required to achieve an optimal density of colonies depend on the inherent plating efficiency of the cell line used, the viability and growth phase of the cell suspension culture at the time of plating, as well as the serum concentration in the media. We recommend testing a range of seeding densities between 200 cells/ml and 500 cells/ml for stable and robust cell lines. Based on the density and viability of the cell suspension culture as determined e.g. by haemocytometer count, add the correct amount of cells to the semi-solid media. Invert the tube several times to gently distribute the cells evenly throughout the semi-solid media. Please note; It is crucial that the appropriately treated culture plastics are used. Plate into non TC-treated culture dishes. Dispense 2ml per well of a Molecular Devices PetriWell-6 Plate or 9ml of media per Molecular Devices PetriWell-1 Plate or 100mm tissue culture dish. This may be achieved using a 10ml pipette or by pouring. Tilt the culture dish gently to ensure even distribution. The incubation time required for CHO cells in semi-solid media requires precautions to keep the semi-solid media well hydrated. To this end fill areas of the plate between wells with ca. 2-4ml of sterile water. Verify the presence of growing colonies at a suitable time point using a light microscope Molecular Devices > 8 of 10
3.2 CHO-plating for adherent colonies The correct density of colonies in the culture dish is key to the success of colony selection and the automated picking process. It is therefore crucial to thoroughly optimise the seeding densities. For picking on Molecular Devices ClonePix systems the optimal density of adherent CHO colonies is ca. 25 colonies per 1ml of semi-solid media. For the procedure described below plating efficiency is high. We recommend evaluation of a range of seeding densities (ca. 25 cells/ml and 50 cells/ml). Please note; It is crucial that the appropriately treated culture plastics are used. Plate into TC-treated culture dishes in the usual liquid media supplemented with serum (as per standard protocol). Plate cells in 2ml media per well of a Molecular Devices PetriWell-6 Plate or 9ml of media per Molecular Devices PetriWell-1 Plate or 100mm tissue culture dish. Place in an incubator at 37 C, 5% CO 2 over night and allow cells to adhere. Verify good adherence using a light microscope. Prepare semi-solid media as described in section 1 above and supplement with the same amount of serum as used in routine liquid culture. Carefully aspirate liquid media and gently overlay with semi-solid media immediately. Dispense 2ml per well of a Molecular Devices PetriWell-6 Plate or 9ml of media per Molecular Devices PetriWell-1 Plate or 100mm tissue culture dish. This may be achieved using a 10ml pipette or by pouring. Tilt the culture dish gently to ensure even distribution of the semi-solid media. Place in an incubator at 37 C, 5% CO 2 for 2-8 10 days to allow colonies to grow. Leave undisturbed for at least 2-8 days. Verify the presence of growing colonies at a suitable time point using a light microscope Molecular Devices > 9 of 10
Contact Details Molecular Devices Queensway, New Milton Hampshire BH25 5NN, UK Tel: +44 (0) 1425 624 600 Fax: +44 (0) 1425 624 700 Web: www.moleculardevices.com/genetix For all technical queries please contact your nearest Customer Support group. Visit www.moleculardevices.com/genetix for latest contact details. Trademarks ClonePix, CloneSelect, CellReporter, HalfBD, 'NRich, SlidePath, Data Arena and Image Arena are trademarks of Molecular Devices (New Milton) Ltd. Copyright 2011 by Molecular Devices (New Milton) Ltd All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form by any means, electronic, mechanical, by photocopying, recording, or otherwise, without the prior written permission of Molecular Devices (New Milton) Ltd. Information furnished by Molecular Devices (New Milton) Ltd is believed to be accurate and reliable; however, no responsibility is assumed by Molecular Devices (New Milton) Ltd, for its use; nor for any infringements of patents or other rights of third parties which may result from its use. No license is granted by implication or otherwise under any patent rights of Molecular Devices (New Milton) Ltd. Revised July 2011 Molecular Devices > 10 of 10