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. 2020 Nov 20;9:e59704. doi: 10.7554/eLife.59704

Figure 4. Lrrc8a KO macrophages are unable to undergo hypotonicity-induced regulatory volume decrease (RVD).

(A) Generation of LRRC8A conditional allele. LRRC8A is found on mouse chromosome two and consists of four exons. Untranslated sequences are represented by black boxes, and coding sequences by grey boxes. Exon three contains the vast majority of coding sequence and was flanked by loxP sites in two sequential steps, first integrating the 5’ LoxP by CRISPR-Cas9 (scissors) mediated double strand break and the supply of a homology flanked ssODN repair template containing the loxP site (grey triangle). This 5’ fl background was then bred to establish a colony and the process repeated to integrate the second 3’ loxP on this background. At each step, integration of loxP was confirmed by PCR and Sanger sequencing. Finally crossing with a Cre driver knocked into the Cx3cr1 locus results in recombinase mediated excision of Exon 3. (B) Western blot of LRRC8A from wild-type (WT) or Lrrc8a knockout (KO) bone-marrow-derived macrophages (BMDMs) and peritoneal macrophages (Mϕ) (n = 3). (C) Regulatory volume decrease measured by calcein fluorescence in WT or Lrrc8a KO BMDMs incubated in a hypotonic buffer (117 mOsm kg−1) (n = 4–5). (D) Area under the curve (AUC) analysis of (C) (n = 4–5). (E) Representative phase contrast images of WT or Lrrc8a KO BMDMs incubated in a hypotonic buffer (117 mOsm kg−1) at indicated time points (n = 3, Scale = 20 µm). ***p<0.001 determined by an unpaired t-test. Values shown are mean plus the SEM.

Figure 4—source data 1. source data for data shown in Figure 4C.

Figure 4.

Figure 4—figure supplement 1. Effect of VRAC inhibitors on the RVD response of WT and Lrrc8a KO BMDMs.

Figure 4—figure supplement 1.

Relative cell size of wild-type (WT) or Lrrc8a knockout (KO) murine bone marrow derived macrophages (BMDMs) incubated in isotonic (340 mOsm kg−1) or hypotonic (117 mOsm kg−1) solution, pre-treated with a vehicle control (DMSO) or DCPIB (10 µM) (n = 5). BMDMs were labelled with the fluorescent dye calcein and area of fluorescence was measured over time. These data were generated from the same experiment shown for the Figure 4C–D (vehicle traces). Values shown are mean plus the SEM.