An optimized dissociation protocol for FACS-based isolation of rare cell types from Caenorhabditis elegans L1 larvae

Single-cell isolation and transcriptomic analysis of a specific cell type or tissue offers the possibility of studying cell function and heterogeneity in time-dependent processes with remarkable resolution. The reduced tissue complexity and highly stereotyped development of Caenorhabditis elegans, combined with an extensive genetic toolbox and the ease of growing large tightly synchronized populations makes it an exceptional model organism for the application of such approaches. However, the difficulty to dissociate and isolate single cells from larval stages has been a major constraint to this kind of studies. Here, we describe an improved protocol for dissociation and preparation of single cell suspensions from developmentally synchronized populations of C. elegans L1 larvae. Our protocol has been empirically optimized to allow efficient FACS-based purification of large number of single cells from rare cell types, for subsequent extraction and sequencing of their mRNA.


Description of protocol
Here, we present an optimized protocol for the isolation of large numbers of rare cell types from C. elegans L1 larvae. We built on the knowledge from previous work ( [1][2][3] ), and empirically developed a faster and more robust protocol. This protocol was optimized for the isolation of neuroblasts (Q lineage) as well as epidermal cells (seam cells), but it has also been successfully applied to other cell types such as mesoblasts (M lineage) (Molly Godfrey and Sander van den Heuvel personal communication). Note that even though this protocol was optimized for the isolation of cells from the L1 larval stage, adjustments in the chemical and mechanical treatments steps would in principal make it amenable for the isolation of cells from other larval stages.
To improve the overall output and reduce the time needed for the procedure, we have introduced new steps such as filtration of the L1 arrested larvae sample and filtration of the final cell suspension, as well as the adjustment of the sample washing upon SDS-DTT treatment, which substantially reduces the time of this critical step. In addition, we reformulated the laborious mechanical treatment of the sample by introducing the use of a pellet pestle motor, which allows a more consistent and user-friendly procedure. Finally, we have removed the need for aseptic conditions when growing the C. elegans cultures, a change that had no impact on the quality of the cell sample and the downstream processing. This change not only contributes to a more user-friendly procedure, but also reduces its technical requirements. It should be noted, however, that this protocol was optimized for use of the cell suspension for FACS and subsequent RNA isolation. If cell culturing is intended, the aseptic C. elegans culturing conditions presented by Zhang and colleagues should be taken into consideration.
1. C. elegans culture preparation and growth 1.1. Seed one hundred 90 mm NA22 plates by chunking approximately 100 worms from an ongoing (non-starved) culture. NOTE 1: Use NGM plates seeded with 80 0-10 0 0 μL NA22 bacteria. NOTE 2: Animals to be seeded can be obtained from both synchronous and asynchronous populations. The use of a synchronous population allows maximizing the output of the protocol. NOTE 3: In our experience, 80-100 plates ensure a good cell yield even in situations where there is a higher loss of sample during the steps of this protocol or when chemical and mechanical treatments are less efficient. NOTE: Incubation times shorter than 12 h will result in a suboptimal number of hatched eggs.
2.12. Place two 20 μm nylon filters into a glass funnel and pour the suspension of overnight hatched larvae into a new 50 mL conical tube.
NOTE: If a large number of carcasses and debris are present in the suspension this will result in clogging of the filters and consequent loss of a significant part of the sample. In order to minimize the number larvae lost in this step, two approaches can be taken: 1) split the sample into two 50 mL conical tubes, and filter the content of each tube separately; or 2) resuspend the clogged fraction of the sample and repeat step 2.1 using new filters. IMPORTANT: If L1 arrested larvae are intended to be used proceed to step 3.6, ignoring steps 3.1-3.5, otherwise proceed with step 3.1. NOTE: To maximize the output of the experiment, it is important to minimize the time between the end of the protocol and the sorting of the cells. We typically FACS sort the cells no more than 30 min after the last step of the protocol.

Final considerations regarding quality control and FACS optimization
Efficiency of the protocol and quality of the sample can be assessed by analyzing the cell suspension samples using a hemocytometer and an adequate microscope as described in Zhang et al., 2013. However, the detection of rare and small fluorescent cells using this procedure is very inefficient and hard to implement. We, therefore, recommend using this procedure only at initial trials of the protocol to assess the overall quality of the procedure, i.e., if cell dissociation is occurring as expected. In our hands, the most efficient way of evaluating the presence and quality of rare fluorescent cells is by FACS-based isolation of these cells followed by culture on lectin-coated glass bottom dishes as described in Zhang et al., 2013. Cells should be incubated for 24 to 48 h to allow adhesion to the bottom of the plate before further analysis.
Optimization of the FACS settings is highly dependent on the morphological features of the cell type of interest, quality and spectral features of the fluorescent markers, and the characteristics of the sorting equipment used. It is, therefore, necessarily a trial and error approach, since it is difficult to establish general rules for this procedure. Nevertheless, the following recommendations can contribute to minimize the optimization time: 50-Use C. elegans strains carrying stably integrated transgenes with high expression of the fluorescent marker(s).
50-When setting up the gating strategy, keep in mind that C. elegans cells are generally small. Flow cytometry facilities are commonly used to develop strategies to sort mammalian cells which are much larger in size. 50-In each trial, sort different sub-populations that could fit the criteria for the desired cells separately. This can decrease the number of trials needed to find the correct population of cells. 50-In parallel to the evaluation based on the cell culture mentioned above, a fraction of the sample can be resorted to access its purity and efficiency of the sorting.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.